Compositions and methods for wt1 specific immunotherapy

ABSTRACT

Compositions and methods for the therapy of malignant diseases, such as leukemia and cancer, are disclosed. The compositions comprise one or more of a WT1 polynucleotide, a WT1 polypeptide, an antigen-presenting cell presenting a WT1 polypeptide, an antibody that specifically binds to a WT1 polypeptide; or a T cell that specifically reacts with a WT1 polypeptide. Such compositions may be used, for example, for the prevention and treatment of metastatic diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/427,717, filed Apr. 30, 2003, now allowed; which is acontinuation-in-part of U.S. patent application Ser. No. 10/286,333,filed Oct. 30, 2002, now abandoned; which is a continuation-in-part ofU.S. patent application Ser. No. 10/244,830, filed Sep. 16, 2002, nowabandoned; which is a continuation-in-part of U.S. patent applicationSer. No. 10/195,835, filed Jul. 12, 2002, now allowed; which is acontinuation-in-part of U.S. patent application Ser. No. 10/125,635,filed Apr. 16, 2002, now pending; which is a continuation-in-part ofU.S. patent application Ser. No. 10/002,603, filed Oct. 30, 2001, nowpending; which is a continuation-in-part of U.S. patent application Ser.No. 09/938,864, filed Aug. 24, 2001, now abandoned; which applicationsare incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with government support under NIH SBIRPhase I grant number IR43CA81752-01A1. The Government may have certainrights in this invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 210121_(—)610C5_SEQUENCE_LISTING.txt. The textfile is 280 KB, was created on May 21, 2009, and is being submittedelectronically via EFS-Web.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the immunotherapy ofmalignant diseases such as leukemia and cancers. The invention is morespecifically related to compositions for generating or enhancing animmune response to WT1, and to the use of such compositions forpreventing and/or treating malignant diseases.

2. Description of the Related Art

Cancer and leukemia are significant health problems in the United Statesand throughout the world. Although advances have been made in detectionand treatment of such diseases, no vaccine or other universallysuccessful method for prevention or treatment of cancer and leukemia iscurrently available. Management of the diseases currently relies on acombination of early diagnosis and aggressive treatment, which mayinclude one or more of a variety of treatments such as surgery,radiotherapy, chemotherapy and hormone therapy. The course of treatmentfor a particular cancer is often selected based on a variety ofprognostic parameters, including an analysis of specific tumor markers.However, the use of established markers often leads to a result that isdifficult to interpret, and the high mortality continues to be observedin many cancer patients.

Immunotherapies have the potential to substantially improve cancer andleukemia treatment and survival. Recent data demonstrate that leukemiacan be cured by immunotherapy in the context of bone marrowtransplantation (e.g., donor lymphocyte infusions). Such therapies mayinvolve the generation or enhancement of an immune response to atumor-associated antigen (TAA). However, to date relatively few TAAs areknown and the generation of an immune response against such antigenshas, with rare exception, not been shown to be therapeuticallybeneficial.

Accordingly, there is a need in the art for improved methods forleukemia and cancer prevention and therapy. The present inventionfulfills these needs and further provides other related advantages.

BRIEF SUMMARY OF THE INVENTION

Briefly stated, this invention provides compositions and methods for thediagnosis and therapy of diseases such as leukemia and cancer. In oneaspect, the present invention provides polypeptides comprising animmunogenic portion of a native WT1, or a variant thereof that differsin one or more substitutions, deletions, additions and/or insertionssuch that the ability of the variant to react with antigen-specificantisera and/or T-cell lines or clones is not substantially diminished.Within certain embodiments of the present invention, the polypeptidecomprises at least an immunogenic portion of WT1 wherein the immunogenicportion is contained within amino acids 2-281 of WT1. Within certainembodiments, the polypeptide comprises no more than 16 consecutive aminoacid residues of a native WT1 polypeptide. Within other embodiments, thepolypeptide comprises an immunogenic portion of amino acid residues1-174 of a native WT1 polypeptide or a variant thereof, wherein thepolypeptide comprises no more than 16 consecutive amino acid residuespresent within amino acids 175 to 449 of the native WT1 polypeptide. Theimmunogenic portion preferably binds to an MHC class I and/or class IImolecule. Within certain embodiments, the polypeptide comprises asequence selected from the group consisting of (a) sequences recited inany one or more of Tables II-XLVI, (b) variants of the foregoingsequences that differ in one or more substitutions, deletions, additionsand/or insertions such that the ability of the variant to react withantigen-specific antisera and/or T-cell lines or clones is notsubstantially diminished and (c) mimetics of the polypeptides recitedabove, such that the ability of the mimetic to react withantigen-specific antisera and/or T cell lines or clones is notsubstantially diminished.

Within other embodiments, the polypeptide comprises a sequence selectedfrom the group consisting of (a) ALLPAVPSL (SEQ ID NO:34), GATLKGVAA(SEQ ID NO:88), CMTWNQMNL (SEQ ID NOs: 49 and 258), SCLESQPTI (SEQ IDNOs: 199 and 296), SCLESQPAI (SEQ ID NO:198), NLYQMTSQL (SEQ ID NOs: 147and 284), ALLPAVSSL (SEQ ID NOs: 35 and 255), RMFPNAPYL (SEQ ID NOs: 185and 293), VLDFAPPGA (SEQ ID NO:241), VLDFAPPGAS (SEQ ID NO:411), SEQ IDNOs: 414-450, ALLPAVPSL (SEQ ID NO:451) (b) variants of the foregoingsequences that differ in one or more substitutions, deletions, additionsand/or insertions such that the ability of the variant to react withantigen-specific antisera and/or T-cell lines or clones is notsubstantially diminished and (c) mimetics of the polypeptides recitedabove, such that the ability of the mimetic to react withantigen-specific antisera and/or T cell lines or clones is notsubstantially diminished. Mimetics may comprises amino acids incombination with one or more amino acid mimetics or may be entirelynonpeptide mimetics.

Within further aspects, the present invention provides polypeptidescomprising a variant of an immunogenic portion of a WT1 protein, whereinthe variant differs from the immunogenic portion due to substitutions atbetween 1 and 3 amino acid positions within the immunogenic portion suchthat the ability of the variant to react with antigen-specific antiseraand/or T-cell lines or clones is not substantially reduced or isenhanced relative to a native WT1 protein.

The present invention further provides WT1 polynucleotides that encode aWT1 polypeptide as described above.

Within other aspects, the present invention provides pharmaceuticalcompositions and vaccines. Pharmaceutical compositions may comprise apolypeptide or mimetic as described above and/or one or more of (i) aWT1 polynucleotide; (ii) an antibody or antigen-binding fragment thereofthat specifically binds to a WT1 polypeptide; (iii) a T cell thatspecifically reacts with a WT1 polypeptide or (iv) an antigen-presentingcell that expresses a WT1 polypeptide, in combination with apharmaceutically acceptable carrier or excipient. Vaccines comprise apolypeptide as described above and/or one or more of (i) a WT1polynucleotide, (ii) an antigen-presenting cell that expresses a WT1polypeptide or (iii) an anti-idiotypic antibody, and a non-specificimmune response enhancer. Within certain embodiments, less than 23consecutive amino acid residues, preferably less than 17 amino acidresidues, of a native WT1 polypeptide are present within a WT1polypeptide employed within such pharmaceutical compositions andvaccines. The immune response enhancer may be an adjuvant. Preferably,an immune response enhancer enhances a T cell response.

The present invention further provides methods for enhancing or inducingan immune response in a patient, comprising administering to a patient apharmaceutical composition or vaccine as described above. In certainembodiments, the patient is a human.

The present invention further provides methods for inhibiting thedevelopment of a malignant disease in a patient, comprisingadministering to a patient a pharmaceutical composition or vaccine asdescribed above. Malignant diseases include, but are not limited toleukemias (e.g., acute myeloid, acute lymphocytic and chronic myeloid)and cancers (e.g., breast, lung, thyroid or gastrointestinal cancer or amelanoma). The patient may, but need not, be afflicted with themalignant disease, and the administration of the pharmaceuticalcomposition or vaccine may inhibit the onset of such a disease, or mayinhibit progression and/or metastasis of an existing disease.

The present invention further provides, within other aspects, methodsfor removing cells expressing WT1 from bone marrow and/or peripheralblood or fractions thereof, comprising contacting bone marrow,peripheral blood or a fraction of bone marrow or peripheral blood with Tcells that specifically react with a WT1 polypeptide, wherein the stepof contacting is performed under conditions and for a time sufficient topermit the removal of WT1 positive cells to less than 10%, preferablyless than 5% and more preferably less than 1%, of the number of myeloidor lymphatic cells in the bone marrow, peripheral blood or fraction.Bone marrow, peripheral blood and fractions may be obtained from apatient afflicted with a disease associated with WT1 expression, or maybe obtained from a human or non-human mammal not afflicted with such adisease.

Within related aspects, the present invention provides methods forinhibiting the development of a malignant disease in a patient,comprising administering to a patient bone marrow, peripheral blood or afraction of bone marrow or peripheral blood prepared as described above.Such bone marrow, peripheral blood or fractions may be autologous, ormay be derived from a related or unrelated human or non-human animal(e.g., syngeneic or allogeneic).

In other aspects, the present invention provides methods for stimulating(or priming) and/or expanding T cells, comprising contacting T cellswith a WT1 polypeptide under conditions and for a time sufficient topermit the stimulation and/or expansion of T cells. Such T cells may beautologous, allogeneic, syngeneic or unrelated WT1-specific T cells, andmay be stimulated in vitro or in vivo. Expanded T cells may, withincertain embodiments, be present within bone marrow, peripheral blood ora fraction of bone marrow or peripheral blood, and may (but need not) beclonal. Within certain embodiments, T cells may be present in a mammalduring stimulation and/or expansion. WT1-specific T cells may be used,for example, within donor lymphocyte infusions.

Within related aspects, methods are provided for inhibiting thedevelopment of a malignant disease in a patient, comprisingadministering to a patient T cells prepared as described above. Such Tcells may, within certain embodiments, be autologous, syngeneic orallogeneic.

The present invention further provides, within other aspects, methodsfor monitoring the effectiveness of an immunization or therapy for amalignant disease associated with WT1 expression in a patient. Suchmethods are based on monitoring antibody, CD4+ T cell and/or CD8+ T cellresponses in the patient. Within certain such aspects, a method maycomprise the steps of: (a) incubating a first biological sample with oneor more of: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1polypeptide; or (iii) an antigen presenting cell that expresses a WT1polypeptide, wherein the first biological sample is obtained from apatient prior to a therapy or immunization, and wherein the incubationis performed under conditions and for a time sufficient to allowimmunocomplexes to form; (b) detecting immunocomplexes formed betweenthe WT1 polypeptide and antibodies in the biological sample thatspecifically bind to the WT1 polypeptide; (c) repeating steps (a) and(b) using a second biological sample obtained from the same patientfollowing therapy or immunization; and (d) comparing the number ofimmunocomplexes detected in the first and second biological samples, andtherefrom monitoring the effectiveness of the therapy or immunization inthe patient.

Within certain embodiments of the above methods, the step of detectingcomprises (a) incubating the immunocomplexes with a detection reagentthat is capable of binding to the immunocomplexes, wherein the detectionreagent comprises a reporter group, (b) removing unbound detectionreagent, and (c) detecting the presence or absence of the reportergroup. The detection reagent may comprise, for example, a secondantibody, or antigen-binding fragment thereof, capable of binding to theantibodies that specifically bind to the WT1 polypeptide or a moleculesuch as Protein A. Within other embodiments, a reporter group is boundto the WT1 polypeptide, and the step of detecting comprises removingunbound WT1 polypeptide and subsequently detecting the presence orabsence of the reporter group.

Within further aspects, methods for monitoring the effectiveness of animmunization or therapy for a malignant disease associated with WT1expression in a patient may comprise the steps of: (a) incubating afirst biological sample with one or more of: (i) a WT1 polypeptide; (ii)a polynucleotide encoding a WT1 polypeptide; or (iii) an antigenpresenting cell that expresses a WT1 polypeptide, wherein the biologicalsample comprises CD4+ and/or CD8+ T cells and is obtained from a patientprior to a therapy or immunization, and wherein the incubation isperformed under conditions and for a time sufficient to allow specificactivation, proliferation and/or lysis of T cells; (b) detecting anamount of activation, proliferation and/or lysis of the T cells; (c)repeating steps (a) and (b) using a second biological sample comprisingCD4+ and/or CD8+ T cells, wherein the second biological sample isobtained from the same patient following therapy or immunization; and(d) comparing the amount of activation, proliferation and/or lysis of Tcells in the first and second biological samples, and therefrommonitoring the effectiveness of the therapy or immunization in thepatient.

The present invention further provides methods for inhibiting thedevelopment of a malignant disease associated with WT1 expression in apatient, comprising the steps of: (a) incubating CD4⁺ and/or CD8+ Tcells isolated from a patient with one or more of: (i) a WT1polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii)an antigen presenting cell that expresses a WT1 polypeptide, such thatthe T cells proliferate; and (b) administering to the patient aneffective amount of the proliferated T cells, and therefrom inhibitingthe development of a malignant disease in the patient. Within certainembodiments, the step of incubating the T cells may be repeated one ormore times.

Within other aspects, the present invention provides methods forinhibiting the development of a malignant disease associated with WT1expression in a patient, comprising the steps of: (a) incubating CD4⁺and/or CD8+ T cells isolated from a patient with one or more of: (i) aWT1 polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or(iii) an antigen presenting cell that expresses a WT1 polypeptide, suchthat the T cells proliferate; (b) cloning one or more cells thatproliferated; and (c) administering to the patient an effective amountof the cloned T cells.

Within other aspects, methods are provided for determining the presenceor absence of a malignant disease associated with WT1 expression in apatient, comprising the steps of: (a) incubating CD4⁺ and/or CD8+ Tcells isolated from a patient with one or more of: (i) a WT1polypeptide; (ii) a polynucleotide encoding a WT1 polypeptide; or (iii)an antigen presenting cell that expresses a WT1 polypeptide; and (b)detecting the presence or absence of specific activation of the T cells,therefrom determining the presence or absence of a malignant diseaseassociated with WT1 expression. Within certain embodiments, the step ofdetecting comprises detecting the presence or absence of proliferationof the T cells.

Within further aspects, the present invention provides methods fordetermining the presence or absence of a malignant disease associatedwith WT1 expression in a patient, comprising the steps of: (a)incubating a biological sample obtained from a patient with one or moreof: (i) a WT1 polypeptide; (ii) a polynucleotide encoding a WT1polypeptide; or (iii) an antigen presenting cell that expresses a WT1polypeptide, wherein the incubation is performed under conditions andfor a time sufficient to allow immunocomplexes to form; and (b)detecting immunocomplexes formed between the WT1 polypeptide andantibodies in the biological sample that specifically bind to the WT1polypeptide; and therefrom determining the presence or absence of amalignant disease associated with WT1 expression.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a comparison of the mouse (MO) and human (HU) WT1 proteinsequences (SEQ ID NOS: 320 and 319 respectively).

FIG. 2 is a Western blot illustrating the detection of WT1 specificantibodies in patients with hematological malignancy (AML). Lane 1 showsmolecular weight markers; lane 2 shows a positive control (WT1 positivehuman leukemia cell line immunoprecipitated with a WT1 specificantibody); lane 3 shows a negative control (WT1 positive cell lineimmunoprecipitated with mouse sera); and lane 4 shows a WT1 positivecell line immunoprecipitated with sera of a patient with AML. For lanes2-4, the immunoprecipitate was separated by gel electrophoresis andprobed with a WT1 specific antibody.

FIG. 3 is a Western blot illustrating the detection of a WT1 specificantibody response in B6 mice immunized with TRAMP-C, a WT1 positivetumor cell line. Lanes 1, 3 and 5 show molecular weight markers, andlanes 2, 4 and 6 show a WT1 specific positive control (N180, Santa CruzBiotechnology, polypeptide spanning 180 amino acids of the N-terminalregion of the WT1 protein, migrating on the Western blot at 52 kD). Theprimary antibody used was WT180 in lane 2, sera of non-immunized B6 micein lane 4 and sera of the immunized B6 mice in lane 6.

FIG. 4 is a Western blot illustrating the detection of WT1 specificantibodies in mice immunized with representative WT1 peptides. Lanes 1,3 and 5 show molecular weight markers and lanes 2, 4 and 6 show a WT1specific positive control (N180, Santa Cruz Biotechnology, polypeptidespanning 180 amino acids of the N-terminal region of the WT1 protein,migrating on the Western blot at 52 kD). The primary antibody used wasWT180 in lane 2, sera of non-immunized B6 mice in lane 4 and sera of theimmunized B6 mice in lane 6.

FIGS. 5A to 5C are graphs illustrating the stimulation of proliferativeT cell responses in mice immunized with representative WT1 peptides.Thymidine incorporation assays were performed using one T cell line andtwo different clones, as indicated, and results were expressed as cpm.Controls indicated on the x axis were no antigen (No Ag) and B6/media;antigens used were p6-22 human (p1), p117-139 (p2) or p244-262 human(p3).

FIGS. 6A and 6B are histograms illustrating the stimulation ofproliferative T cell responses in mice immunized with representative WT1peptides. Three weeks after the third immunization, spleen cells of micethat had been inoculated with Vaccine A or Vaccine B were cultured withmedium alone (medium) or spleen cells and medium (B6/no antigen), B6spleen cells pulsed with the peptides p6-22 (p6), p117-139 (p117),p244-262 (p244) (Vaccine A; FIG. 6A) or p287-301 (p287), p299-313(p299), p421-435 (p421) (Vaccine B; FIG. 6B) and spleen cells pulsedwith an irrelevant control peptide (irrelevant peptide) at 25 ug/ml andwere assayed after 96 hr for proliferation by (³H) thymidineincorporation. Bars represent the stimulation index (SI), which iscalculated as the mean of the experimental wells divided by the mean ofthe control (B6 spleen cells with no antigen).

FIGS. 7A-7D are histograms illustrating the generation of proliferativeT-cell lines and clones specific for p117-139 and p6-22. Following invivo immunization, the initial three in vitro stimulations (IVS) werecarried out using all three peptides of Vaccine A or B, respectively.Subsequent IVS were carried out as single peptide stimulations usingonly the two relevant peptides p117-139 and p6-22. Clones were derivedfrom both the p6-22 and p117-139 specific T cell lines, as indicated. Tcells were cultured with medium alone (medium) or spleen cells andmedium (B6/no antigen), B6 spleen cells pulsed with the peptides p6-22(p6), p117-139 (p117) or an irrelevant control peptide (irrelevantpeptide) at 25 ug/ml and were assayed after 96 hr for proliferation by(³H) thymidine incorporation. Bars represent the stimulation index (SI),which is calculated as the mean of the experimental wells divided by themean of the control (B6 spleen cells with no antigen).

FIGS. 8A and 8B present the results of TSITES Analysis of human WT1 (SEQID NO:319) for peptides that have the potential to elicit Th responses.Regions indicated by “A” are AMPHI midpoints of blocks, “R” indicatesresidues matching the Rothbard/‘Taylor motif, “D” indicates residuesmatching the IAd motif, and ‘d’ indicates residues matching the IEdmotif.

FIGS. 9A and 9B are graphs illustrating the elicitation of WT1peptide-specific CTL in mice immunized with WT1 peptides. FIG. 9Aillustrates the lysis of target cells by allogeneic cell lines and FIG.9B shows the lysis of peptide coated cell lines. In each case, the %lysis (as determined by standard chromium release assays) is shown atthree indicated effector:target ratios. Results are provided forlymphoma cells (LSTRA and E10), as well as E10+p235-243 (E10+P235). E10cells are also referred to herein as EL-4 cells.

FIGS. 10A-10D are graphs illustrating the elicitation of WT1 specificCTL, which kill WT1 positive tumor cell lines but do not kill WT1negative cell lines, following vaccination of B6 mice with WT1 peptideP117. FIG. 10A illustrates that T-cells of non-immunized B6 mice do notkill WT1 positive tumor cell lines.

FIG. 10B illustrates the lysis of the target cells by allogeneic celllines. FIGS. 10C and 10D demonstrate the lysis of WT1 positive tumorcell lines, as compared to WT1 negative cell lines in two differentexperiments. In addition, FIGS. 10C and 10D show the lysis ofpeptide-coated cell lines (WT1 negative cell line E10 coated with therelevant WT1 peptide P117) In each case, the % lysis (as determined bystandard chromium release assays) is shown at three indicatedeffector:target ratios. Results are provided for lymphoma cells (E10),prostate cancer cells (TRAMP-C), a transformed fibroblast cell line(BLK-SV40), as well as E10+p117.

FIGS. 11A and 11B are histograms illustrating the ability ofrepresentative peptide P117-139 specific CTL to lyse WT1 positive tumorcells. Three weeks after the third immunization, spleen cells of micethat had been inoculated with the peptides p235-243 or p117-139 werestimulated in vitro with the relevant peptide and tested for ability tolyse targets incubated with WT1 peptides as well as WT1 positive andnegative tumor cells. The bars represent the mean % specific lysis inchromium release assays performed in triplicate with an E:T ratio of25:1. FIG. 11A shows the cytotoxic activity of the p235-243 specific Tcell line against the WT1 negative cell line EL-4 (EL-4, WT1 negative);EL-4 pulsed with the relevant (used for immunization as well as forrestimulation) peptide p235-243 (EL-4+p235); EL-4 pulsed with theirrelevant peptides p117-139 (EL-4+p117), p126-134 (EL-4+p126) orp130-138 (EL-4+p130) and the WT1 positive tumor cells BLK-SV40(BLK-SV40, WT1 positive) and TRAMP-C (TRAMP-C, WT1 positive), asindicated. FIG. 11B shows cytotoxic activity of the p117-139 specific Tcell line against EL-4; EL-4 pulsed with the relevant peptide P117-139(EL-4+p117) and EL-4 pulsed with the irrelevant peptides p123-131(EL-4+p123), or p128-136 (EL-4+p128); BLK-SV40 and TRAMP-C, asindicated.

FIGS. 12A and 12B are histograms illustrating the specificity of lysisof WT1 positive tumor cells, as demonstrated by cold target inhibition.The bars represent the mean % specific lysis in chromium release assaysperformed in triplicate with an E:T ratio of 25:1. FIG. 12A shows thecytotoxic activity of the p117-139 specific T cell line against the WT1negative cell line EL-4 (EL-4, WT1 negative); the WT1 positive tumorcell line TRAMP-C (TRAMP-C, WT1 positive); TRAMP-C cells incubated witha ten-fold excess (compared to the hot target) of EL-4 cells pulsed withthe relevant peptide p117-139 (TRAMP-C+p117 cold target) without ⁵¹Crlabeling and TRAMP-C cells incubated with EL-4 pulsed with an irrelevantpeptide without ⁵¹Cr labeling (TRAMP-C+irrelevant cold target), asindicated. FIG. 12B shows the cytotoxic activity of the p117-139specific T cell line against the WT1 negative cell line EL-4 (EL-4, WT1negative); the WT1 positive tumor cell line BLK-SV40 (BLK-SV40, WT1positive); BLK-SV40 cells incubated with the relevant cold target(BLK-SV40+p117 cold target) and BLK-SV40 cells incubated with theirrelevant cold target (BLK-SV40+irrelevant cold target), as indicated.

FIGS. 13A-13C are histograms depicting an evaluation of the 9mer CTLepitope within p117-139. The p117-139 tumor specific CTL line was testedagainst peptides within aa117-139 containing or lacking an appropriateH-2^(b) class I binding motif and following restimulation with p126-134or p130-138. The bars represent the mean % specific lysis in chromiumrelease assays performed in triplicate with an E:T ratio of 25:1. FIG.13A shows the cytotoxic activity of the p117-139 specific T cell lineagainst the WT1 negative cell line EL-4 (EL-4, WT1 negative) and EL-4cells pulsed with the peptides p117-139 (EL-4+p117), p119-127(EL-4+p119), p120-128 (EL-4+p120), p123-131 (EL-4+p123), p126-134(EL-4+p126), p128-136 (EL-4+p128), and p130-138 (EL-4+p130). FIG. 13Bshows the cytotoxic activity of the CTL line after restimulation withp126-134 against the WT1 negative cell line EL-4, EL-4 cells pulsed withp117-139 (EL-4+p117), p126-134 (EL-4+p126) and the WT1 positive tumorcell line TRAMP-C. FIG. 13C shows the cytotoxic activity of the CTL lineafter restimulation with p130-138 against EL-4, EL-4 cells pulsed withp117-139 (EL-4+p117), p130-138 (EL-4+p130) and the WT1 positive tumorcell line TRAMP-C.

FIG. 14 depicts serum antibody reactivity to WT1 in 63 patients withAML. Reactivity of serum antibody to WT1/N-terminus protein wasevaluated by ELISA in patients with AML. The first and second lanesrepresent the positive and negative controls, respectively. The firstand second lanes represent the positive and negative controls,respectively. Commercially obtained WT1 specific antibody WT180 was usedfor the positive control. The next 63 lanes represent results using serafrom each individual patient. The OD values depicted were from ELISAusing a 1:500 serum dilution. The figure includes cumulative data from 3separate experiments.

FIG. 15 depicts serum antibody reactivity to WT1 proteins and controlproteins in 2 patients with AML. Reactivity of serum antibody toWT1/full-length, WT1N-terminus, TRX and Ra12 proteins was evaluated byELISA in 2 patients with AML. The OD values depicted were from ELISAusing a 1:500 serum dilution. AML-1 and AML-2 denote serum from 2 of theindividual patients in FIG. 1 with demonstrated antibody reactivity toWT1/full-length. The WT1 full-length protein was expressed as a fusionprotein with Ra12. The WT1/N-terminus protein was expressed as a fusionprotein with TRX. The control Ra12 and TRX proteins were purified in asimilar manner. The results confirm that the serum antibody reactivityagainst the WT1 fusion proteins is directed against the WT1 portions ofthe protein.

FIG. 16 depicts serum antibody reactivity to WT1 in 81 patients withCML. Reactivity of serum antibody to WT1/full-length protein wasevaluated by ELISA in patients with AML. The first and second lanesrepresent the positive and negative controls, respectively. Commerciallyobtained WT1 specific antibody WT180 was used for the positive control.The next 81 lanes represent results using sera from each individualpatient. The OD values depicted were from ELISA using a 1:500 serumdilution. The figure includes cumulative data from 3 separateexperiments.

FIG. 17 depicts serum antibody reactivity to WT1 proteins and controlproteins in 2 patients with CML. Reactivity of serum antibody toWT1/full-length, WT1/N-terminus, TRX and Ra12 proteins was evaluated byELISA in 2 patients with CML. The OD values depicted were from ELISAusing a 1:500 serum dilution. CML-1 and CML-2 denote serum from 2 of theindividual patients in FIG. 3 with demonstrated antibody reactivity toWT1/full-length. The WT1/full-length protein was expressed as a fusionprotein with Ra12. The WT1/N-terminus protein was expressed as a fusionprotein with TRX. The control Ra12 and TRX proteins were purified in asimilar manner. The results confirm that the serum antibody reactivityagainst the WT1 fusion proteins is directed against the WT1 portions ofthe protein.

FIG. 18 provides the characteristics of the recombinant WT1 proteinsused for serological analysis.

FIGS. 19A1-19E3 is a bar graph depicting the antibody responses in miceelicited by vaccination with different doses of WT1 protein.

FIGS. 20A and B is a bar graph of the proliferative T-cell responses inmice immunized with WT1 protein.

FIG. 21 is a photograph of human DC, examined by fluorescent microscopy,expressing WT1 following adeno WT1 and Vaccinia WT1 infection.

FIG. 22 is a photograph that demonstrates that WT1 expression in humanDC is reproducible following adeno WT1 infection and is not induced by acontrol Adeno infection.

FIG. 23 is a graph of an IFN-gamma ELISPOT assay showing that WT1 wholegene in vitro priming elicits WT1 specific T-cell responses.

FIG. 24 shows amino acids 2-281 (SEQ ID NO:461) of the WT1 protein andthe cDNA encoding these amino acid residues (SEQ ID NO:460). Thistruncated WT1 protein is referred to as WT1-F.

DETAILED DESCRIPTION OF THE INVENTION

U.S. patents, U.S. patent application publications, U.S. patentapplications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, are incorporated herein by reference, intheir entirety.

As noted above, the present invention is generally directed tocompositions and methods for the immunotherapy and diagnosis ofmalignant diseases. The compositions described herein may include WT1polypeptides, WT1 polynucleotides, antigen-presenting cells (APC, e.g.,dendritic cells) that express a WT1 polypeptide, agents such asantibodies that bind to a WT1 polypeptide and/or immune system cells(e.g., T cells) specific for WT1. WT1 Polypeptides of the presentinvention generally comprise at least a portion of a Wilms Tumor geneproduct (WT1) or a variant thereof. Nucleic acid sequences of thesubject invention generally comprise a DNA or RNA sequence that encodesall or a portion of such a polypeptide, or that is complementary to sucha sequence. Antibodies are generally immune system proteins, orantigen-binding fragments thereof, that are capable of binding to aportion of a WT1 polypeptide. T cells that may be employed within suchcompositions are generally T cells (e.g., CD4⁺ and/or CD8⁺) that arespecific for a WT1 polypeptide. Certain methods described herein furtheremploy antigen-presenting cells that express a WT1 polypeptide asprovided herein.

The present invention is based on the discovery that an immune responseraised against a Wilms Tumor (WT) gene product (e.g., WT1) can provideprophylactic and/or therapeutic benefit for patients afflicted withmalignant diseases characterized by increased WT1 gene expression. Suchdiseases include, but are not limited to, leukemias (e.g., acute myeloidleukemia (AML), chronic myeloid leukemia (CML), acute lymphocyticleukemia (ALL) and childhood ALL), as well as many cancers such as lung,breast, thyroid and gastrointestinal cancers and melanomas. The WT1 genewas originally identified and isolated on the basis of a cytogeneticdeletion at chromosome 11p13 in patients with Wilms' tumor (see Call etal., U.S. Pat. No. 5,350,840). The gene consists of 10 exons and encodesa zinc finger transcription factor, and sequences of mouse and human WT1proteins are provided in FIG. 1 and SEQ ID NOs: 319 and 320.

WT1 Polypeptides

Within the context of the present invention, a WT1 polypeptide is apolypeptide that comprises at least an immunogenic portion of a nativeWT1 (i.e., a WT1 protein expressed by an organism that is notgenetically modified), or a variant thereof, as described herein. A WT1polypeptide may be of any length, provided that it comprises at least animmunogenic portion of a native protein or a variant thereof. In otherwords, a WT1 polypeptide may be an oligopeptide (i.e., consisting of arelatively small number of amino acid residues, such as 8-10 residues,joined by peptide bonds), a full length WT1 protein (e.g., presentwithin a human or non-human animal, such as a mouse) or a polypeptide ofintermediate size. Within certain embodiments, the use of WT1polypeptides that contain a small number of consecutive amino acidresidues of a native WT1 polypeptide is preferred. Such polypeptides arepreferred for certain uses in which the generation of a T cell responseis desired. For example, such a WT1 polypeptide may contain less than23, preferably no more than 18, and more preferably no more than 15consecutive amino acid residues, of a native WT1 polypeptide.Polypeptides comprising nine consecutive amino acid residues of a nativeWT1 polypeptide are generally suitable for such purposes. Additionalsequences derived from the native protein and/or heterologous sequencesmay be present within any WT1 polypeptide, and such sequences may (butneed not) possess further immunogenic or antigenic properties.Polypeptides as provided herein may further be associated (covalently ornoncovalently) with other polypeptide or non-polypeptide compounds.

An “immunogenic portion,” as used herein is a portion of a polypeptidethat is recognized (i.e., specifically bound) by a B-cell and/or T-cellsurface antigen receptor. Certain preferred immunogenic portions bind toan MHC class I or class II molecule. As used herein, an immunogenicportion is said to “bind to” an MHC class I or class II molecule if suchbinding is detectable using any assay known in the art. For example, theability of a polypeptide to bind to MHC class I may be evaluatedindirectly by monitoring the ability to promote incorporation of ¹²⁵Ilabeled β2-microglobulin (β2m) into MHC class I/β2m/peptideheterotrimeric complexes (see Parker et al., J. Immunol. 152:163, 1994).Alternatively, functional peptide competition assays that are known inthe art may be employed. Certain immunogenic portions have one or moreof the sequences recited within one or more of Tables II-XIV.Representative immunogenic portions include, but are not limited to,RDLNALLPAVPSLGGGG (human WT1 residues 6-22; SEQ ID NO:1),PSQASSGQARMFPNAPYLPSCLE (human and mouse WT1 residues 117-139; SEQ IDNOs: 2 and 3 respectively), GATLKGVAAGSSSSVKWTE (human WT1 residues244-262; SEQ ID NO:4), GATLKGVAA (human WT1 residues 244-252; SEQ IDNO:88), CMTWNQMNL (human and mouse WT1 residues 235-243; SEQ ID NOs: 49and 258 respectively), SCLESQPTI (mouse WT1 residues 136-144; SEQ IDNO:296), SCLESQPAI (human WT1 residues 136-144; SEQ ID NO:198),NLYQMTSQL (human and mouse WT1 residues 225-233; SEQ ID NOs: 147 and 284respectively); ALLPAVSSL (mouse WT1 residues 10-18; SEQ ID NO:255);RMFPNAPYL (human and mouse WT1 residues 126-134; SEQ ID NOs: 185 and 293respectively), VLDFAPPGA (human WT1 residues 37-45; SEQ ID NO:241), orVLDFAPPGAS (human WT1 residues 37-46; SEQ ID NO:411). Furtherimmunogenic portions are provided in SEQ ID NOs:414-451. Furtherimmunogenic portions are provided herein, and others may generally beidentified using well known techniques, such as those summarized inPaul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) andreferences cited therein. Representative techniques for identifyingimmunogenic portions include screening polypeptides for the ability toreact with antigen-specific antisera and/or T-cell lines or clones. Animmunogenic portion of a native WT1 polypeptide is a portion that reactswith such antisera and/or T-cells at a level that is not substantiallyless than the reactivity of the full length WT1 (e.g., in an ELISAand/or T-cell reactivity assay). In other words, an immunogenic portionmay react within such assays at a level that is similar to or greaterthan the reactivity of the full length polypeptide. Such screens maygenerally be performed using methods well known to those of ordinaryskill in the art, such as those described in Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.

Alternatively, immunogenic portions may be identified using computeranalysis, such as the Tsites program (see Rothbard and Taylor, EMBO J.7:93-100, 1988; Deavin et al., Mol. Immunol. 33:145-155, 1996), whichsearches for peptide motifs that have the potential to elicit Thresponses. CTL peptides with motifs appropriate for binding to murineand human class I or class II MHC may be identified according to BIMAS(Parker et al., J. Immunol. 152:163, 1994) and other HLA peptide bindingprediction analyses. Alternatively, immungenic portions that bind to aparticular MHC molecule can be identified by using defined peptidebinding motifs such as those described in Rammensee et al.,Immunogenetics 41:178-228, 1995. To confirm peptide binding to murineand human class I or class II MHC molecules, peptide binding assaysknown in the art may be used. To confirm immunogenicity, a peptide maybe tested using an HLA A2 or other transgenic mouse model and/or an invitro stimulation assay using dendritic cells, fibroblasts or peripheralblood cells.

As noted above, a composition may comprise a variant of a native WT1protein. A polypeptide “variant,” as used herein, is a polypeptide thatdiffers from a native polypeptide in one or more substitutions,deletions, additions and/or insertions, such that the immunogenicity ofthe polypeptide is retained (i.e., the ability of the variant to reactwith antigen-specific antisera and/or T-cell lines or clones is notsubstantially diminished relative to the native polypeptide). In otherwords, the ability of a variant to react with antigen-specific antiseraand/or T-cell lines or clones may be enhanced or unchanged, relative tothe native polypeptide, or may be diminished by less than 50%, andpreferably less than 20%, relative to the native polypeptide. In oneembodiment the ability of a variant to react with antigen-specificantisera and/or T-cell lines or clones may be diminished by less than10%, 5%, 4%, 3%, 2%, 1%, or 0.5%, relative to the native polypeptide.Such variants may generally be identified by modifying one of the abovepolypeptide sequences and evaluating the reactivity of the modifiedpolypeptide with antisera and/or T-cells as described herein. In oneembodiment of the present invention, a variant may be identified byevaluating its ability to bind to a human or a muring HLA molecule. Inone preferred embodiment, a variant polypeptide has a modification suchthat the ability of the variant polypeptide to bind to a class I orclass II MHC molecule, for example HLA-A2, HLA-A24, HLA-A1, HLA-A3,HLA-A68, HLA-A11, HLA-A31, HLA-A33, HLA-B14, HLA-B40, HLA-B60, HLA-B62,HLA-B7, HLA-B8, HLA-B27, HLA-B3501, HLA-B37, HLA-B38, HLA-B39, HLA-B44,HLA-B51, HLA-B52, HLA-B58, HLA-CW03, HLA-CW04, HLA-CW06, or HLA-CW07, isincreased relative to that of a wild type (unmodified) WT1 polypeptide.In a further embodiment, the N-terminal portion of WT1 from amino acids1-281 is modified such that the ability of any number of polypeptidestherein that can bind to HLA-A2, HLA-A24, HLA-A1, HLA-A3, HLA-A68,HLA-A11, HLA-A31, HLA-A33, HLA-B14, HLA-B40, HLA-B60, HLA-B62, HLA-B7,HLA-B8, HLA-B27, HLA-B3501, HLA-B37, HLA-B38, HLA-B39, HLA-B44, HLA-B51,HLA-B52, HLA-B58, HLA-CW03, HLA-CW04, HLA-CW06, or HLA-CW07 is increasedrelative to that of the wild type (unmodified) WT1 polypeptide sequence.In a further embodiment, a polypeptide comprising at least oneimmunogenic portion (epitope) is modified such that its ability to bindto an MHC molecule, such as HLA-A2, HLA-A24, HLA-A1, HLA-A3, HLA-A68,HLA-A11, HLA-A31, HLA-A33, HLA-B14, HLA-B40, HLA-B60, HLA-B62, HLA-B7,HLA-B8, HLA-B27, HLA-B3501, HLA-B37, HLA-B38, HLA-B39, HLA-B44, HLA-B51,HLA-B52, HLA-B58, HLA-CW03, HLA-CW04, HLA-CW06, or HLA-CW07 isincreased. Example 30 describes illustrative variants that can begenerated. The skilled artisan would readily recognize that these areillustrative variants and that other variants can be generated in asimilar manner for peptides that bind to HLA molecules other thanHLA-A2. In a further embodiment, the ability of the variant polypeptideto bind to a HLA molecule is increased by at least 2 fold, preferably atleast 3 fold, 4 fold, or 5 fold relative to that of a native WT1polypeptide. It has been found, within the context of the presentinvention, that a relatively small number of substitutions (e.g., 1 to3) within an immunogenic portion of a WT1 polypeptide may serve toenhance the ability of the polypeptide to elicit an immune response.Suitable substitutions may generally be identified by using computerprograms, as described above, and the effect confirmed based on thereactivity of the modified polypeptide with antisera and/or T-cells asdescribed herein. Accordingly, within certain preferred embodiments, aWT1 polypeptide comprises a variant in which 1 to 3 amino acid resideswithin an immunogenic portion are substituted such that the ability toreact with antigen-specific antisera and/or T-cell lines or clones isstatistically greater than that for the unmodified polypeptide. Suchsubstitutions are preferably located within an MHC binding site of thepolypeptide, which may be identified as described above. Preferredsubstitutions allow increased binding to MHC class I or class IImolecules.

Certain variants contain conservative substitutions. A “conservativesubstitution” is one in which an amino acid is substituted for anotheramino acid that has similar properties, such that one skilled in the artof peptide chemistry would expect the secondary structure andhydropathic nature of the polypeptide to be substantially unchanged.Amino acid substitutions may generally be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. Variants may also (oralternatively) be modified by, for example, the deletion or addition ofamino acids that have minimal influence on the immunogenicity, secondarystructure and hydropathic nature of the polypeptide.

In a preferred embodiment, a variant polypeptide of the WT1 N-terminus(amino acids 1-249) is constructed, wherein the variant polypeptide iscapable of binding to an antibody that recognizes full-length WT1 and/orWT1 N-terminus polypeptide. A non-limiting example of an antibody isanti WT1 antibody WT180 (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.).

As noted above, WT1 polypeptides may be conjugated to a signal (orleader) sequence at the N-terminal end of the protein whichco-translationally or post-translationally directs transfer of theprotein. A polypeptide may also, or alternatively, be conjugated to alinker or other sequence for ease of synthesis, purification oridentification of the polypeptide (e.g., poly-His), or to enhancebinding of the polypeptide to a solid support. For example, apolypeptide may be conjugated to an immunoglobulin Fc region.

WT1 polypeptides may be prepared using any of a variety of well knowntechniques. Recombinant polypeptides encoded by a WT1 polynucleotide asdescribed herein may be readily prepared from the polynucleotide. Ingeneral, any of a variety of expression vectors known to those ofordinary skill in the art may be employed to express recombinant WT1polypeptides. Expression may be achieved in any appropriate host cellthat has been transformed or transfected with an expression vectorcontaining a DNA molecule that encodes a recombinant polypeptide.Suitable host cells include prokaryotes, yeast and higher eukaryoticcells. Preferably, the host cells employed are E. coli, yeast or amammalian cell line such as COS or CHO. Supernatants from suitablehost/vector systems which secrete recombinant protein or polypeptideinto culture media may be first concentrated using a commerciallyavailable filter. The concentrate may then be applied to a suitablepurification matrix such as an affinity matrix or an ion exchange resin.Finally, one or more reverse phase HPLC steps can be employed to furtherpurify a recombinant polypeptide. Such techniques may be used to preparenative polypeptides or variants thereof. For example, polynucleotidesthat encode a variant of a native polypeptide may generally be preparedusing standard mutagenesis techniques, such as oligonucleotide-directedsite-specific mutagenesis, and sections of the DNA sequence may beremoved to permit preparation of truncated polypeptides.

Certain portions and other variants may also be generated by syntheticmeans, using techniques well known to those of ordinary skill in theart. For example, polypeptides having fewer than about 500 amino acids,preferably fewer than about 100 amino acids, and more preferably fewerthan about 50 amino acids, may be synthesized. Polypeptides may besynthesized using any of the commercially available solid-phasetechniques, such as the Merrifield solid-phase synthesis method, whereamino acids are sequentially added to a growing amino acid chain. SeeMerrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment forautomated synthesis of polypeptides is commercially available fromsuppliers such as Applied BioSystems, Inc. (Foster City, Calif.), andmay be operated according to the manufacturer's instructions.

In general, polypeptides and polynucleotides as described herein areisolated. An “isolated” polypeptide or polynucleotide is one that isremoved from its original environment. For example, anaturally-occurring protein is isolated if it is separated from some orall of the coexisting materials in the natural system. Preferably, suchpolypeptides are at least about 90% pure, more preferably at least about95% pure and most preferably at least about 99% pure. A polynucleotideis considered to be isolated if, for example, it is cloned into a vectorthat is not a part of the natural environment.

Within further aspects, the present invention provides mimetics of WT1polypeptides. Such mimetics may comprise amino acids linked to one ormore amino acid mimetics (i.e., one or more amino acids within the WT1protein may be replaced by an amino acid mimetic) or may be entirelynonpeptide mimetics. An amino acid mimetic is a compound that isconformationally similar to an amino acid such that it can besubstituted for an amino acid within a WT1 polypeptide withoutsubstantially diminishing the ability to react with antigen-specificantisera and/or T cell lines or clones. A nonpeptide mimetic is acompound that does not contain amino acids, and that has an overallconformation that is similar to a WT1 polypeptide such that the abilityof the mimetic to react with WT1-specific antisera and/or T cell linesor clones is not substantially diminished relative to the ability of aWT1 polypeptide. Such mimetics may be designed based on standardtechniques (e.g., nuclear magnetic resonance and computationaltechniques) that evaluate the three dimensional structure of a peptidesequence. Mimetics may be designed where one or more of the side chainfunctionalities of the WT1 polypeptide are replaced by groups that donot necessarily have the same size or volume, but have similar chemicaland/or physical properties which produce similar biological responses.It should be understood that, within embodiments described herein, amimetic may be substituted for a WT1 polypeptide.

Within other illustrative embodiments, a polypeptide may be a fusionpolypeptide that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence, such as a known tumor protein. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the polypeptide or to enable the polypeptide to betargeted to desired intracellular compartments. Still further fusionpartners include affinity tags, which facilitate purification of thepolypeptide.

Fusion polypeptides may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion polypeptide isexpressed as a recombinant polypeptide, allowing the production ofincreased levels, relative to a non-fused polypeptide, in an expressionsystem. Briefly, DNA sequences encoding the polypeptide components maybe assembled separately, and ligated into an appropriate expressionvector. The 3′ end of the DNA sequence encoding one polypeptidecomponent is ligated, with or without a peptide linker, to the 5′ end ofa DNA sequence encoding the second polypeptide component so that thereading frames of the sequences are in phase. This permits translationinto a single fusion polypeptide that retains the biological activity ofboth component polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion polypeptideusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

The fusion polypeptide can comprise a polypeptide as described hereintogether with an unrelated immunogenic protein, such as an immunogenicprotein capable of eliciting a recall response. Examples of suchproteins include tetanus, tuberculosis and hepatitis proteins (see, forexample, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

In one preferred embodiment, the immunological fusion partner is derivedfrom a Mycobacterium sp., such as a Mycobacterium tuberculosis-derivedRa12 fragment. Ra12 compositions and methods for their use in enhancingthe expression and/or immunogenicity of heterologouspolynucleotide/polypeptide sequences is described in U.S. PatentApplication 60/158,585, the disclosure of which is incorporated hereinby reference in its entirety. Briefly, Ra12 refers to a polynucleotideregion that is a subsequence of a Mycobacterium tuberculosis MTB32Anucleic acid. MTB32A is a serine protease of 32 KD molecular weightencoded by a gene in virulent and avirulent strains of M. tuberculosis.The nucleotide sequence and amino acid sequence of MTB32A have beendescribed (for example, U.S. Patent Application 60/158,585; see also,Skeiky et al., Infection and Immun. (1999) 67:3998-4007, incorporatedherein by reference). C-terminal fragments of the MTB32A coding sequenceexpress at high levels and remain as soluble polypeptides throughout thepurification process. Moreover, Ra12 may enhance the immunogenicity ofheterologous immunogenic polypeptides with which it is fused. Onepreferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragmentcorresponding to amino acid residues 192 to 323 of MTB32A. Otherpreferred Ra12 polynucleotides generally comprise at least about 15consecutive nucleotides, at least about 30 nucleotides, at least about60 nucleotides, at least about 100 nucleotides, at least about 200nucleotides, or at least about 300 nucleotides that encode a portion ofa Ra12 polypeptide. Ra12 polynucleotides may comprise a native sequence(i.e., an endogenous sequence that encodes a Ra12 polypeptide or aportion thereof) or may comprise a variant of such a sequence. Ra12polynucleotide variants may contain one or more substitutions,additions, deletions and/or insertions such that the biological activityof the encoded fusion polypeptide is not substantially diminished,relative to a fusion polypeptide comprising a native Ra12 polypeptide.Variants preferably exhibit at least about 70% identity, more preferablyat least about 80% identity and most preferably at least about 90%identity to a polynucleotide sequence that encodes a native Ra12polypeptide or a portion thereof.

Within other preferred embodiments, an immunological fusion partner isderived from protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionpolypeptide. A repeat portion is found in the C-terminal region startingat residue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

Within another illustrative embodiment the fusion partner comprises atwin arginine translocation (TAT) signal peptide from the TorA signalpeptide in E. coli on the N-terminus; see J. Mol. Microbiol. (2000)2(2): 179-189; Journal of Bacteriology, January 2001 p604-610 Vol 183,No 2; Journal of Biochemistry Vol 276, Mar. 16, 2001 pp 8159-8164),hereby incorporated herein in its entirety.

Yet another illustrative embodiment involves fusion polypeptides, andthe polynucleotides encoding them, wherein the fusion partner comprisesa targeting signal capable of directing a polypeptide to theendosomal/lysosomal compartment, as described in U.S. Pat. No.5,633,234. An immunogenic polypeptide of the invention, when fused withthis targeting signal, will associate more efficiently with MHC class IImolecules and thereby provide enhanced in vivo stimulation of CD4⁺T-cells specific for the polypeptide.

The invention provides truncated forms of WT1 polypeptides that can berecombinantly expressed in E. coli without the addition of a fusionpartner. Examples of these truncated forms are shown in SEQ IDNOs:342-346, and are encoded by polynucleotides shown in SEQ IDNOs:337-341. In variations of these truncations, the first 76 aminoacids of WT1 can be fused to the C-terminus of the protein, creating arecombinant protein that is easier to express in E. coli. Other hosts inaddition to E. coli can also be used, such as, for example, B.megaterium. The protein can further be prepared without a histidine tag.

In other embodiments, different subunits can be made and fused togetherin an order which differs from that of native WT1. In addition, fusionscan be made with, for example, Ra12. Exemplary fusion proteins are shownin SEQ ID NOs: 332-336 and can be encoded by polynucleotides shown inSEQ ID NOs: 327-331.

WT1 Polynucleotides

Any polynucleotide that encodes a WT1 polypeptide as described herein isa WT1 polynucleotide encompassed by the present invention. Suchpolynucleotides may be single-stranded (coding or antisense) ordouble-stranded, and may be DNA (genomic, cDNA or synthetic) or RNAmolecules. Additional coding or non-coding sequences may, but need not,be present within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials.

WT1 polynucleotides may encode a native WT1 protein, or may encode avariant of WT1 as described herein. Polynucleotide variants may containone or more substitutions, additions, deletions and/or insertions suchthat the immunogenicity of the encoded polypeptide is not diminished,relative to a native WT1 protein. The effect on the immunogenicity ofthe encoded polypeptide may generally be assessed as described herein.Preferred variants contain nucleotide substitutions, deletions,insertions and/or additions at no more than 20%, preferably at no morethan 10%, of the nucleotide positions that encode an immunogenic portionof a native WT1 sequence. Certain variants are substantially homologousto a native gene, or a portion thereof. Such polynucleotide variants arecapable of hybridizing under moderately stringent conditions to anaturally occurring DNA sequence encoding a WT1 polypeptide (or acomplementary sequence). Suitable moderately stringent conditionsinclude prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH8.0); hybridizing at 50° C.-65° C., 5×SSC, overnight; followed bywashing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSCcontaining 0.1% SDS). Such hybridizing DNA sequences are also within thescope of this invention.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a WT1 polypeptide. Some of these polynucleotidesbear minimal homology to the nucleotide sequence of any native gene.Nonetheless, polynucleotides that vary due to differences in codon usageare specifically contemplated by the present invention.

Therefore, according to another aspect of the present invention,polynucleotide compositions are provided that comprise some or all of apolynucleotide sequence set forth herein, complements of apolynucleotide sequence set forth herein, and degenerate variants of apolynucleotide sequence set forth herein. In certain preferredembodiments, the polynucleotide sequences set forth herein encodeimmunogenic polypeptides, as described above.

Once an immunogenic portion of WT1 is identified, as described above, aWT1 polynucleotide may be prepared using any of a variety of techniques.For example, a WT1 polynucleotide may be amplified from cDNA preparedfrom cells that express WT1. Such polynucleotides may be amplified viapolymerase chain reaction (PCR). For this approach, sequence-specificprimers may be designed based on the sequence of the immunogenic portionand may be purchased or synthesized. For example, suitable primers forPCR amplification of a human WT1 gene include: first step—P118:1434-1414: 5′ GAG AGT CAG ACT TGA AAG CAGT 3′ (SEQ ID NO:5) and P135: 5′CTG AGC dC AGC AAA TGG GC 3′ (SEQ ID NO:6); second step—P136: 5′ GAG CATGCA TGG GCT CCG ACG TGC GGG 3′ (SEQ ID NO:7) and P137: 5′ GGG GTA CCCACT GAA CGG TCC CCG A 3′ (SEQ ID NO:8). Primers for PCR amplification ofa mouse WT1 gene include: first step—P138: 5′ TCC GAG CCG CAC CTC ATG 3′(SEQ ID NO:9) and P139: 5′ GCC TGG GAT GCT GGA CTG 3′ (SEQ ID NO:10),second step—P140: 5′ GAG CAT GCG ATG GGT TCC GAC GTG CGG 3′ (SEQ IDNO:11) and P141: 5′ GGG GTA CCT CAA AGC GCC ACG TGG AGT TT 3′ (SEQ IDNO:12).

An amplified portion may then be used to isolate a full length gene froma human genomic DNA library or from a suitable cDNA library, using wellknown techniques. Alternatively, a full length gene can be constructedfrom multiple PCR fragments. WT1 polynucleotides may also be prepared bysynthesizing oligonucleotide components, and ligating componentstogether to generate the complete polynucleotide.

WT1 polynucleotides may also be synthesized by any method known in theart, including chemical synthesis (e.g., solid phase phosphoramiditechemical synthesis). Modifications in a polynucleotide sequence may alsobe introduced using standard mutagenesis techniques, such asoligonucleotide-directed site-specific mutagenesis (see Adelman et al.,DNA 2:183, 1983). Alternatively, RNA molecules may be generated by invitro or in vivo transcription of DNA sequences encoding a WT1polypeptide, provided that the DNA is incorporated into a vector with asuitable RNA polymerase promoter (such as T7 or SP6). Certain portionsmay be used to prepare an encoded polypeptide, as described herein. Inaddition, or alternatively, a portion may be administered to a patientsuch that the encoded polypeptide is generated in vivo (e.g., bytransfecting antigen-presenting cells such as dendritic cells with acDNA construct encoding a WT1 polypeptide, and administering thetransfected cells to the patient).

Polynucleotides that encode a WT1 polypeptide may generally be used forproduction of the polypeptide, in vitro or in vivo. WT1 polynucleotidesthat are complementary to a coding sequence (i.e., antisensepolynucleotides) may also be used as a probe or to inhibit WT1expression. cDNA constructs that can be transcribed into antisense RNAmay also be introduced into cells of tissues to facilitate theproduction of antisense RNA.

Any polynucleotide may be further modified to increase stability invivo. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends; the use ofphosphorothioate or 2′ O-methyl rather than phosphodiesterase linkagesin the backbone; and/or the inclusion of nontraditional bases such asinosine, queosine and wybutosine, as well as acetyl-methyl-, thio- andother modified forms of adenine, cytidine, guanine, thymine and uridine.

Nucleotide sequences as described herein may be joined to a variety ofother nucleotide sequences using established recombinant DNA techniques.

For example, a polynucleotide may be cloned into any of a variety ofcloning vectors, including plasmids, phagemids, lambda phage derivativesand cosmids. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors and sequencing vectors. Ingeneral, a vector will contain an origin of replication functional in atleast one organism, convenient restriction endonuclease sites and one ormore selectable markers. Other elements will depend upon the desireduse, and will be apparent to those of ordinary skill in the art. Inparticular, one embodiment of the invention comprises expression vectorswhich incorporate the nucleic acid molecules of the invention, inoperable linkage (i.e., “operably linked”) to an expression controlsequence (promoter). Illustrative vectors include, but are not limited,those described herein in Examples 20 and 40. Construction of vacciniavirus or attenuated viral vectors is well within the skill of the art,as is the transformation or transfection of cells, to produce eukaryoticcell lines, or prokaryotic cell strains which encode the molecule ofinterest. Exemplary of the host cells which can be employed in thisfashion are COS cells, CHO cells, yeast cells, insect cells (e.g.,Spodoptera frugiperda or Sf-9 cells), NIH 3T3 cells, and so forth.Prokaryotic cells, such as E. coli and other bacteria may also be used.

Within certain embodiments, polynucleotides may be formulated so as topermit entry into a cell of a mammal, and expression therein. Suchformulations are particularly useful for therapeutic purposes, asdescribed below. Those of ordinary skill in the art will appreciate thatthere are many ways to achieve expression of a polynucleotide in atarget cell, and any suitable method may be employed. For example, apolynucleotide may be incorporated into a viral vector such as, but notlimited to, adenovirus, adeno-associated virus, retrovirus, or vacciniaor other pox virus (e.g., avian pox virus). Techniques for incorporatingDNA into such vectors are well known to those of ordinary skill in theart. A retroviral vector may additionally transfer or incorporate a genefor a selectable marker (to aid in the identification or selection oftransduced cells) and/or a targeting moiety, such as a gene that encodesa ligand for a receptor on a specific target cell, to render the vectortarget specific. Targeting may also be accomplished using an antibody,by methods known to those of ordinary skill in the art. cDNA constructswithin such a vector may be used, for example, to transfect human oranimal cell lines for use in establishing WT1 positive tumor modelswhich may be used to perform tumor protection and adoptive immunotherapyexperiments to demonstrate tumor or leukemia-growth inhibition or lysisof such cells.

Other therapeutic formulations for polynucleotides include colloidaldispersion systems, such as macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes. A preferredcolloidal system for use as a delivery vehicle in vitro and in vivo is aliposome (i.e., an artificial membrane vesicle). The preparation and useof such systems is well known in the art.

Antibody Compositions, Fragments thereof and Other Binding Agents

According to another aspect, the present invention further providesbinding agents, such as antibodies and antigen-binding fragmentsthereof, that exhibit immunological binding to a WT1 polypeptidedisclosed herein, or to a portion, variant or derivative thereof. Anantibody, or antigen-binding fragment thereof, is said to “specificallybind,” “immunogically bind,” and/or is “immunologically reactive” to aWT1 polypeptide of the invention if it reacts at a detectable level(within, for example, an ELISA assay) with the polypeptide, and does notreact detectably with unrelated polypeptides under similar conditions.

Immunological binding, as used in this context, generally refers to thenon-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. The strength, or affinity of immunological bindinginteractions can be expressed in terms of the dissociation constant(K_(d)) of the interaction, wherein a smaller K_(d) represents a greateraffinity. Immunological binding properties of selected polypeptides canbe quantified using methods well known in the art. One such methodentails measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (K_(on)) and the “off rateconstant” (K_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity, and is thus equal to the dissociation constantK_(d). See, generally, Davies et al. (1990) Annual Rev. Biochem.59:439-473.

An “antigen-binding site,” or “binding portion” of an antibody refers tothe part of the immunoglobulin molecule that participates in antigenbinding. The antigen binding site is formed by amino acid residues ofthe N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”)chains. Three highly divergent stretches within the V regions of theheavy and light chains are referred to as “hypervariable regions” whichare interposed between more conserved flanking stretches known as“framework regions,” or “FRs”. Thus the term “FR” refers to amino acidsequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other in threedimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.”

Binding agents may be further capable of differentiating betweenpatients with and without a WT1-associated cancer, using therepresentative assays provided herein. For example, antibodies or otherbinding agents that bind to a tumor protein will preferably generate asignal indicating the presence of a cancer in at least about 20% ofpatients with the disease, more preferably at least about 30% ofpatients. Alternatively, or in addition, the antibody will generate anegative signal indicating the absence of the disease in at least about90% of individuals without the cancer. To determine whether a bindingagent satisfies this requirement, biological samples (e.g., blood, sera,sputum, urine and/or tumor biopsies) from patients with and without acancer (as determined using standard clinical tests) may be assayed asdescribed herein for the presence of polypeptides that bind to thebinding agent. Preferably, a statistically significant number of sampleswith and without the disease will be assayed. Each binding agent shouldsatisfy the above criteria; however, those of ordinary skill in the artwill recognize that binding agents may be used in combination to improvesensitivity.

Any agent that satisfies the above requirements may be a binding agent.For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for an antigenic polypeptide of interestmay be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

A number of therapeutically useful molecules are known in the art whichcomprise antigen-binding sites that are capable of exhibitingimmunological binding properties of an antibody molecule. Theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′)₂” fragment which comprises bothantigen-binding sites. An “Fv” fragment can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single chain Fv (“sFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Anumber of methods have been described to discern chemical structures forconverting the naturally aggregated—but chemically separated—light andheavy polypeptide chains from an antibody V region into an sFv moleculewhich will fold into a three dimensional structure substantially similarto the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

Each of the above-described molecules includes a heavy chain and a lightchain CDR set, respectively interposed between a heavy chain and a lightchain FR set which provide support to the CDRS and define the spatialrelationship of the CDRs relative to each other. As used herein, theterm “CDR set” refers to the three hypervariable regions of a heavy orlight chain V region. Proceeding from the N-terminus of a heavy or lightchain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) isreferred to herein as a “molecular recognition unit.” Crystallographicanalysis of a number of antigen-antibody complexes has demonstrated thatthe amino acid residues of CDRs form extensive contact with boundantigen, wherein the most extensive antigen contact is with the heavychain CDR3. Thus, the molecular recognition units are primarilyresponsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRS. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated CDRs fused to human constant domains (Winter et al. (1991)Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA86:4220-4224; Shaw et al. (1987) J. Immunol. 138:4534-4538; and Brown etal. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a humansupporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients.

As used herein, the terms “veneered FRs” and “recombinantly veneeredFRs” refer to the selective replacement of FR residues from, e.g., arodent heavy or light chain V region, with human FR residues in order toprovide a xenogeneic molecule comprising an antigen-binding site whichretains substantially all of the native FR polypeptide foldingstructure. Veneering techniques are based on the understanding that theligand binding characteristics of an antigen-binding site are determinedprimarily by the structure and relative disposition of the heavy andlight chain CDR sets within the antigen-binding surface. Davies et al.(1990) Ann. Rev. Biochem. 59:439-473. Thus, antigen binding specificitycan be preserved in a humanized antibody only wherein the CDRstructures, their interaction with each other, and their interactionwith the rest of the V region domains are carefully maintained. By usingveneering techniques, exterior (e.g., solvent-accessible) FR residueswhich are readily encountered by the immune system are selectivelyreplaced with human residues to provide a hybrid molecule that compriseseither a weakly immunogenic, or substantially non-immunogenic veneeredsurface.

The process of veneering makes use of the available sequence data forhuman antibody variable domains compiled by Kabat et al., in Sequencesof Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Healthand Human Services, U.S. Government Printing Office, 1987), updates tothe Kabat database, and other accessible U.S. and foreign databases(both nucleic acid and protein). Solvent accessibilities of V regionamino acids can be deduced from the known three-dimensional structurefor human and murine antibody fragments. There are two general steps inveneering a murine antigen-binding site. Initially, the FRs of thevariable domains of an antibody molecule of interest are compared withcorresponding FR sequences of human variable domains obtained from theabove-identified sources. The most homologous human V regions are thencompared residue by residue to corresponding murine amino acids. Theresidues in the murine FR which differ from the human counterpart arereplaced by the residues present in the human moiety using recombinanttechniques well known in the art. Residue switching is only carried outwith moieties which are at least partially exposed (solvent accessible),and care is exercised in the replacement of amino acid residues whichmay have a significant effect on the tertiary structure of V regiondomains, such as proline, glycine and charged amino acids.

In this manner, the resultant “veneered” murine antigen-binding sitesare thus designed to retain the murine CDR residues, the residuessubstantially adjacent to the CDRs, the residues identified as buried ormostly buried (solvent inaccessible), the residues believed toparticipate in non-covalent (e.g., electrostatic and hydrophobic)contacts between heavy and light chain domains, and the residues fromconserved structural regions of the FRs which are believed to influencethe “canonical” tertiary structures of the CDR loops. These designcriteria are then used to prepare recombinant nucleotide sequences whichcombine the CDRs of both the heavy and light chain of a murineantigen-binding site into human-appearing FRs that can be used totransfect mammalian cells for the expression of recombinant humanantibodies which exhibit the antigen specificity of the murine antibodymolecule.

In another embodiment of the invention, monoclonal antibodies of thepresent invention may be coupled to one or more therapeutic agents.Suitable agents in this regard include radionuclides, differentiationinducers, drugs, toxins, and derivatives thereof. Preferredradionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and²¹²Bi. Preferred drugs include methotrexate, and pyrimidine and purineanalogs. Preferred differentiation inducers include phorbol esters andbutyric acid. Preferred toxins include ricin, abrin, diptheria toxin,cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, andpokeweed antiviral protein.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group. Suitable carriers includeproteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato etal.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat.No. 4,699,784, to Shih et al.). A carrier may also bear an agent bynoncovalent bonding or by encapsulation, such as within a liposomevesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriersspecific for radionuclide agents include radiohalogenated smallmolecules and chelating compounds. For example, U.S. Pat. No. 4,735,792discloses representative radiohalogenated small molecules and theirsynthesis. A radionuclide chelate may be formed from chelating compoundsthat include those containing nitrogen and sulfur atoms as the donoratoms for binding the metal, or metal oxide, radionuclide. For example,U.S. Pat. No. 4,673,562, to Davison et al. discloses representativechelating compounds and their synthesis.

T Cells

Immunotherapeutic compositions may also, or alternatively, comprise Tcells specific for WT1. Such cells may generally be prepared in vitro orex vivo, using standard procedures. For example, T cells may be presentwithin (or isolated from) bone marrow, peripheral blood or a fraction ofbone marrow or peripheral blood of a mammal, such as a patient, using acommercially available cell separation system, such as the CEPRATE™system, available from CellPro Inc., Bothell Wash. (see also U.S. Pat.No. 5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO92/07243). Alternatively, T cells may be derived from related orunrelated humans, non-human animals, cell lines or cultures.

T cells may be stimulated with WT1 polypeptide, polynucleotide encodinga WT1 polypeptide and/or an antigen presenting cell (APC) that expressesa WT1 polypeptide. Such stimulation is performed under conditions andfor a time sufficient to permit the generation of T cells that arespecific for the WT1 polypeptide. Preferably, a WT1 polypeptide orpolynucleotide is present within a delivery vehicle, such as amicrosphere, to facilitate the generation of antigen-specific T cells.Briefly, T cells, which may be isolated from a patient or a related orunrelated donor by routine techniques (such as by FICOLL®/HYPAQUE®density gradient centrifugation of peripheral blood lymphocytes), areincubated with WT1 polypeptide. For example, T cells may be incubated invitro for 2-9 days (typically 4 days) at 37° C. with WT1 polypeptide(e.g., 5 to 25 μg/ml) or cells synthesizing a comparable amount of WT1polypeptide. It may be desirable to incubate a separate aliquot of a Tcell sample in the absence of WT1 polypeptide to serve as a control.

T cells are considered to be specific for a WT1 polypeptide if the Tcells kill target cells coated with a WT1 polypeptide or expressing agene encoding such a polypeptide. T cell specificity may be evaluatedusing any of a variety of standard techniques. For example, within achromium release assay or proliferation assay, a stimulation index ofmore than two fold increase in lysis and/or proliferation, compared tonegative controls, indicates T cell specificity. Such assays may beperformed, for example, as described in Chen et al., Cancer Res.54:1065-1070, 1994. Alternatively, detection of the proliferation of Tcells may be accomplished by a variety of known techniques. For example,T cell proliferation can be detected by measuring an increased rate ofDNA synthesis (e.g., by pulse-labeling cultures of T cells withtritiated thymidine and measuring the amount of tritiated thymidineincorporated into DNA). Other ways to detect T cell proliferationinclude measuring increases in interleukin-2 (IL-2) production, Ca²⁺flux, or dye uptake, such as3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Alternatively,synthesis of lymphokines (such as interferon-gamma) can be measured orthe relative number of T cells that can respond to a WT1 polypeptide maybe quantified. Contact with a WT1 polypeptide (200 ng/ml-100 μg/ml,preferably 100 ng/ml-25 μg/ml) for 3-7 days should result in at least atwo fold increase in proliferation of the T cells and/or contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1, Wiley Interscience (Greene 1998). WT1 specific Tcells may be expanded using standard techniques. Within preferredembodiments, the T cells are derived from a patient or a related orunrelated donor and are administered to the patient followingstimulation and expansion.

T cells that have been activated in response to a WT1 polypeptide,polynucleotide or WT1-expressing APC may be CD4⁺ and/or CD8⁺. Specificactivation of CD4⁺ or CD8⁺ T cells may be detected in a variety of ways.Methods for detecting specific T cell activation include detecting theproliferation of T cells, the production of cytokines (e.g.,lymphokines), or the generation of cytolytic activity (i.e., generationof cytotoxic T cells specific for WT1). For CD4+ T cells, a preferredmethod for detecting specific T cell activation is the detection of theproliferation of T cells. For CD8+ T cells, a preferred method fordetecting specific T cell activation is the detection of the generationof cytolytic activity.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to the WT1 polypeptide, polynucleotide or APC can be expandedin number either in vitro or in vivo. Proliferation of such T cells invitro may be accomplished in a variety of ways. For example, the T cellscan be re-exposed to WT1 polypeptide, with or without the addition of Tcell growth factors, such as interleukin-2, and/or stimulator cells thatsynthesize a WT1 polypeptide. The addition of stimulator cells ispreferred where generating CD8⁺ T cell responses. T cells can be grownto large numbers in vitro with retention of specificity in response tointermittent restimulation with WT1 polypeptide. Briefly, for theprimary in vitro stimulation (IVS), large numbers of lymphocytes (e.g.,greater than 4×10⁷) may be placed in flasks with media containing humanserum. WT1 polypeptide (e.g., peptide at 10 μg/ml) may be addeddirectly, along with tetanus toxoid (e.g., 5 μg/ml). The flasks may thenbe incubated (e.g., 37° C. for 7 days). For a second IVS, T cells arethen harvested and placed in new flasks with 2−3×10⁷ irradiatedperipheral blood mononuclear cells. WT1 polypeptide (e.g., 10 μg/ml) isadded directly. The flasks are incubated at 37° C. for 7 days. On day 2and day 4 after the second IVS, 2-5 units of interleukin-2 (IL-2) may beadded. For a third IVS, the T cells may be placed in wells andstimulated with the individual's own EBV transformed B cells coated withthe peptide. IL-2 may be added on days 2 and 4 of each cycle. As soon asthe cells are shown to be specific cytotoxic T cells, they may beexpanded using a 10 day stimulation cycle with higher IL-2 (20 units) ondays 2, 4 and 6.

Alternatively, one or more T cells that proliferate in the presence ofWT1 polypeptide can be expanded in number by cloning. Methods forcloning cells are well known in the art, and include limiting dilution.Responder T cells may be purified from the peripheral blood ofsensitized patients by density gradient centrifugation and sheep redcell rosetting and established in culture by stimulating with thenominal antigen in the presence of irradiated autologous filler cells.In order to generate CD4⁺ T cell lines, WT1 polypeptide is used as theantigenic stimulus and autologous peripheral blood lymphocytes (PBL) orlymphoblastoid cell lines (LCL) immortalized by infection with EpsteinBarr virus are used as antigen presenting cells. In order to generateCD8⁺ T cell lines, autologous antigen-presenting cells transfected withan expression vector which produces WT1 polypeptide may be used asstimulator cells. Established T cell lines may be cloned 2-4 daysfollowing antigen stimulation by plating stimulated T cells at afrequency of 0.5 cells per well in 96-well flat-bottom plates with 1×10⁶irradiated PBL or LCL cells and recombinant interleukin-2 (rIL2) (50U/ml). Wells with established clonal growth may be identified atapproximately 2-3 weeks after initial plating and restimulated withappropriate antigen in the presence of autologous antigen-presentingcells, then subsequently expanded by the addition of low doses of rIL2(10 U/ml) 2-3 days following antigen stimulation. T cell clones may bemaintained in 24-well plates by periodic restimulation with antigen andrIL2 approximately every two weeks.

Within certain embodiments, allogeneic T-cells may be primed (i.e.,sensitized to WT1) in vivo and/or in vitro. Such priming may be achievedby contacting T cells with a WT1 polypeptide, a polynucleotide encodingsuch a polypeptide or a cell producing such a polypeptide underconditions and for a time sufficient to permit the priming of T cells.In general, T cells are considered to be primed if, for example, contactwith a WT1 polypeptide results in proliferation and/or activation of theT cells, as measured by standard proliferation, chromium release and/orcytokine release assays as described herein. A stimulation index of morethan two fold increase in proliferation or lysis, and more than threefold increase in the level of cytokine, compared to negative controls,indicates T-cell specificity. Cells primed in vitro may be employed, forexample, within a bone marrow transplantation or as donor lymphocyteinfusion.

T cells specific for WT1 can kill cells that express WT1 protein.Introduction of genes encoding T-cell receptor (TCR) chains for WT1 areused as a means to quantitatively and qualitatively improve responses toWT1 bearing leukemia and cancer cells. Vaccines to increase the numberof T cells that can react to WT1 positive cells are one method oftargeting WT1 bearing cells. T cell therapy with T cells specific forWT1 is another method. An alternative method is to introduce the TCRchains specific for WT1 into T cells or other cells with lyticpotential. In a suitable embodiment, the TCR alpha and beta chains arecloned out from a WT1 specific T cell line and used for adoptive T celltherapy, such as described in WO96/30516, incorporated herein byreference.

T Cell Receptor Compositions

The T cell receptor (TCR) consists of 2 different, highly variablepolypeptide chains, termed the T-cell receptor α and β chains, that arelinked by a disulfide bond (Janeway, Travers, Walport. Immunobiology.Fourth Ed., 148-159. Elsevier Science Ltd/Garland Publishing. 1999). Theα/β heterodimer complexes with the invariant CD3 chains at the cellmembrane. This complex recognizes specific antigenic peptides bound toMHC molecules. The enormous diversity of TCR specificities is generatedmuch like immunoglobulin diversity, through somatic gene rearrangement.The β chain genes contain over 50 variable (V), 2 diversity (D), over 10joining (J) segments, and 2 constant region segments (C). The α chaingenes contain over 70 V segments, and over 60 J segments but no Dsegments, as well as one C segment. During T cell development in thethymus, the D to J gene rearrangement of the β chain occurs, followed bythe V gene segment rearrangement to the DJ. This functional VDJβ exon istranscribed and spliced to join to a Cβ. For the α chain, a Vα genesegment rearranges to a Jα gene segment to create the functional exonthat is then transcribed and spliced to the Cox. Diversity is furtherincreased during the recombination process by the random addition of Pand N-nucleotides between the V, D, and J segments of the chain andbetween the V and J segments in the α chain (Janeway, Travers, Walport.Immunobiology. Fourth Ed., 98 and 150. Elsevier Science Ltd/GarlandPublishing. 1999).

The present invention, in another aspect, provides TCRs specific for apolypeptide disclosed herein, or for a variant or derivative thereof. Inaccordance with the present invention, polynucleotide and amino acidsequences are provided for the V-J or V-D-J junctional regions or partsthereof for the alpha and beta chains of the T-cell receptor whichrecognize tumor polypeptides described herein. In general, this aspectof the invention relates to T-cell receptors which recognize or bindtumor polypeptides presented in the context of MHC. In a preferredembodiment the tumor antigens recognized by the T-cell receptorscomprise a polypeptide of the present invention. For example, cDNAencoding a TCR specific for a WT1 peptide can be isolated from T cellsspecific for a tumor polypeptide using standard molecular biological andrecombinant DNA techniques.

This invention further includes the T-cell receptors or analogs thereofhaving substantially the same function or activity as the T-cellreceptors of this invention which recognize or bind tumor polypeptides.Such receptors include, but are not limited to, a fragment of thereceptor, or a substitution, addition or deletion mutant of a T-cellreceptor provided herein. This invention also encompasses polypeptidesor peptides that are substantially homologous to the T-cell receptorsprovided herein or that retain substantially the same activity. The term“analog” includes any protein or polypeptide having an amino acidresidue sequence substantially identical to the T-cell receptorsprovided herein in which one or more residues, preferably no more than 5residues, more preferably no more than 25 residues have beenconservatively substituted with a functionally similar residue and whichdisplays the functional aspects of the T-cell receptor as describedherein.

The present invention further provides for suitable mammalian hostcells, for example, non-specific T cells, that are transfected with apolynucleotide encoding TCRs specific for a polypeptide describedherein, thereby rendering the host cell specific for the polypeptide.The α and β chains of the TCR may be contained on separate expressionvectors or alternatively, on a single expression vector that alsocontains an internal ribosome entry site (IRES) for cap-independenttranslation of the gene downstream of the IRES. Said host cellsexpressing TCRs specific for the polypeptide may be used, for example,for adoptive immunotherapy of WT1-associated cancer as discussed furtherbelow.

In further aspects of the present invention, cloned TCRs specific for apolypeptide recited herein may be used in a kit for the diagnosis ofWT1-associated cancer. For example, the nucleic acid sequence orportions thereof, of tumor-specific TCRs can be used as probes orprimers for the detection of expression of the rearranged genes encodingthe specific TCR in a biological sample. Therefore, the presentinvention further provides for an assay for detecting messenger RNA orDNA encoding the TCR specific for a polypeptide.

Peptide-MHC Tetrameric Complexes

The present invention, in another aspect, provides peptide-MHCtetrameric complexes (tetramers) specific for T cells that recognize apolypeptide disclosed herein, or for a variant or derivative thereof. Inone embodiment, tetramers may be used in the detection of WT1 specificT-cells. Tetramers may be used in monitoring WT1 specific immuneresponses, early detection of WT1 associated malignancies and formonitoring minimal residual disease. Tetramer staining is typicallycarried out with flow cytometric analysis and can be used to identifygroups within a patient population suffering from a WT1 associateddisease at a higher risk for relapse or disease progression.

Pharmaceutical Compositions

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide, T-cell, TCR, and/orantibody compositions disclosed herein in pharmaceutically-acceptablecarriers for administration to a cell or an animal, either alone, or incombination with one or more other modalities of therapy.

It will be understood that, if desired, a composition as disclosedherein may be administered in combination with other agents as well,such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact, there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

Therefore, in another aspect of the present invention, pharmaceuticalcompositions are provided comprising one or more of the polynucleotide,polypeptide, antibody, TCR, and/or T-cell compositions described hereinin combination with a physiologically acceptable carrier. In certainpreferred embodiments, the pharmaceutical compositions of the inventioncomprise immunogenic polynucleotide and/or polypeptide compositions ofthe invention for use in prophylactic and therapeutic vaccineapplications. Vaccine preparation is generally described in, forexample, M. F. Powell and M. J. Newman, eds., “Vaccine Design (thesubunit and adjuvant approach),” Plenum Press (NY, 1995). Generally,such compositions will comprise one or more polynucleotide and/orpolypeptide compositions of the present invention in combination withone or more immunostimulants.

It will be apparent that any of the pharmaceutical compositionsdescribed herein can contain pharmaceutically acceptable salts of thepolynucleotides and polypeptides of the invention. Such salts can beprepared, for example, from pharmaceutically acceptable non-toxic bases,including organic bases (e.g., salts of primary, secondary and tertiaryamines and basic amino acids) and inorganic bases (e.g., sodium,potassium, lithium, ammonium, calcium and magnesium salts).

In another embodiment, illustrative immunogenic compositions, e.g.,vaccine compositions, of the present invention comprise DNA encoding oneor more of the polypeptides as described above, such that thepolypeptide is generated in situ. As noted above, the polynucleotide maybe administered within any of a variety of delivery systems known tothose of ordinary skill in the art. Indeed, numerous gene deliverytechniques are well known in the art, such as those described byRolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, andreferences cited therein. Appropriate polynucleotide expression systemswill, of course, contain the necessary regulatory DNA regulatorysequences for expression in a patient (such as a suitable promoter andterminating signal). Alternatively, bacterial delivery systems mayinvolve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface or secretes such an epitope.

Therefore, in certain embodiments, polynucleotides encoding immunogenicpolypeptides described herein are introduced into suitable mammalianhost cells for expression using any of a number of known viral-basedsystems. In one illustrative embodiment, retroviruses provide aconvenient and effective platform for gene delivery systems. A selectednucleotide sequence encoding a polypeptide of the present invention canbe inserted into a vector and packaged in retroviral particles usingtechniques known in the art. The recombinant virus can then be isolatedand delivered to a subject. A number of illustrative retroviral systemshave been described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman(1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993)Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin(1993) Cur. Opin. Genet. Develop. 3:102-109.

In addition, a number of illustrative adenovirus-based systems have alsobeen described. Unlike retroviruses which integrate into the hostgenome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476).

Various adeno-associated virus (AAV) vector systems have also beendeveloped for polynucleotide delivery. AAV vectors can be readilyconstructed using techniques well known in the art. See, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

Additional viral vectors useful for delivering the polynucleotidesencoding polypeptides of the present invention by gene transfer includethose derived from the pox family of viruses, such as vaccinia virus andavian poxvirus. By way of example, vaccinia virus recombinantsexpressing the novel molecules can be constructed as follows. The DNAencoding a polypeptide is first inserted into an appropriate vector sothat it is adjacent to a vaccinia promoter and flanking vaccinia DNAsequences, such as the sequence encoding thymidine kinase (TK). Thisvector is then used to transfect cells which are simultaneously infectedwith vaccinia. Homologous recombination serves to insert the vacciniapromoter plus the gene encoding the polypeptide of interest into theviral genome. The resulting TK.sup.(−) recombinant can be selected byculturing the cells in the presence of 5-bromodeoxyuridine and pickingviral plaques resistant thereto.

A vaccinia-based infection/transfection system can be conveniently usedto provide for inducible, transient expression or coexpression of one ormore polypeptides described herein in host cells of an organism. In thisparticular system, cells are first infected in vitro with a vacciniavirus recombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the polynucleotide or polynucleotides of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAwhich is then translated into polypeptide by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990)87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986)83:8122-8126.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the coding sequences of interest.Recombinant avipoxviruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.A number of pox viruses have been developed as live viral vectors forthe expression of heterologous proteins (Cepko et al., Cell 37:1053-1062(1984); Morin et al., Proc. Natl. Acad. Sci. USA 84:4626-4630 (1987);Lowe et al., Proc. Natl. Acad. Sci. USA, 84:3896-3900 (1987); Panicali &Paoletti, Proc. Natl. Acad. Sci. USA, 79:4927-4931 (1982); Machett etal., Proc. Natl. Acad. Sci. USA, 79:7415-7419 (1982)). Representativefowlpox and swinepox virus are available through the ATCC underaccession numbers VR-229 and VR-363, respectively. A recombinantvaccinia—CEA is available through the ATCC under accession numberVR2323. Other illustrative viral vectors also include, but are notlimited to, those described by Therion Biologics (Cambridge, Mass.,USA), for example, in U.S. Pat. Nos. 6,051,410, 5,858,726, 5,656,465,5,804,196, 5,747,324, 6,319,496, 6,165,460.

Any of a number of alphavirus vectors can also be used for delivery ofpolynucleotide compositions of the present invention, such as thosevectors described in U.S. Pat. Nos. 5,843,723; 6,015,686; 6,008,035 and6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE)can also be used, illustrative examples of which can be found in U.S.Pat. Nos. 5,505,947 and 5,643,576.

Moreover, molecular conjugate vectors, such as the adenovirus chimericvectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, canalso be used for gene delivery under the invention.

Additional illustrative information on these and other known viral-baseddelivery systems can be found, for example, in Fisher-Hoch et al., Proc.Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y. Acad.Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat.Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993.

As would be readily appreciated by the skilled artisan, any number ofadditional components may be present in a DNA or retroviral vectorexpressing a WT1 polypeptide or any portion thereof, as describedherein. For example, an expression vector for delivery of apolynucleotide or peptide of the present invention may include anynumber of a variety of costimulatory molecules, including, but notlimited to CD28, B7-1, ICAM-1, and LFA-3. A delivery vector may alsoinclude any number of cytokines, for example IFN-γ, GM-CSF, or IL-2. Inone illustrative embodiment, a recombinant viral vector, e.g. a vacciniaor fowlpox vector, includes B7-1, ICAM-1, and LFA-3.

The present invention also comprises the use of any combination of theDNA and/or viral vectors described herein for use in the treatment ofmalignancies associated with the expression of WT1. In one illustrativeembodiment, a recombinant vaccinia viral vector is administered to ananimal or human patient afflicted with a WT1-associated malignancy,followed by administration of a recombinant fowlpox vector. In anotherembodiment of the present invention, the recombinant fowlpox isadminstered twice following administration of the vaccinia vector (e.g.a prime/boost/boost vaccination regimen).

In certain embodiments, a polynucleotide may be integrated into thegenome of a target cell. This integration may be in the specificlocation and orientation via homologous recombination (gene replacement)or it may be integrated in a random, non-specific location (geneaugmentation). In yet further embodiments, the polynucleotide may bestably maintained in the cell as a separate, episomal segment of DNA.Such polynucleotide segments or “episomes” encode sequences sufficientto permit maintenance and replication independent of or insynchronization with the host cell cycle. The manner in which theexpression construct is delivered to a cell and where in the cell thepolynucleotide remains is dependent on the type of expression constructemployed.

In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

In still another embodiment, a composition of the present invention canbe delivered via a particle bombardment approach, many of which havebeen described. In one illustrative example, gas-driven particleacceleration can be achieved with devices such as those manufactured byPowderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc.(Madison, Wis.), some examples of which are described in U.S. Pat. Nos.5,846,796; 6,010,478; 5,865,796; 5,584,807; and EP Patent No. 0500 799.This approach offers a needle-free delivery approach wherein a drypowder formulation of microscopic particles, such as polynucleotide orpolypeptide particles, are accelerated to high speed within a helium gasjet generated by a hand held device, propelling the particles into atarget tissue of interest.

In a related embodiment, other devices and methods that may be usefulfor gas-driven needle-less injection of compositions of the presentinvention include those provided by Bioject, Inc. (Portland, Oreg.),some examples of which are described in U.S. Pat. Nos. 4,790,824;5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

According to another embodiment, the pharmaceutical compositionsdescribed herein will comprise one or more immunostimulants in additionto the immunogenic polynucleotide, polypeptide, antibody, T-cell, TCR,and/or APC compositions of this invention. An immunostimulant refers toessentially any substance that enhances or potentiates an immuneresponse (antibody and/or cell-mediated) to an exogenous antigen. Onepreferred type of immunostimulant comprises an adjuvant. Many adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Certain adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham,Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, -12, and other like growth factors, may alsobe used as adjuvants.

Within certain embodiments of the invention, the adjuvant composition ispreferably one that induces an immune response predominantly of the Th1type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

Certain preferred adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A(MPL®), preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL™),together with an aluminum salt. MPL® adjuvants are available from CorixaCorporation (Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727;4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (inwhich the CpG dinucleotide is unmethylated) also induce a predominantlyTh1 response. Such oligonucleotides are well known and are described,for example, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200and 5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOM®s (saponins,cholesterol and phospholipids). Furthermore, the saponins may beformulated together with a polyoxyethylene ether or ester, in either anon-particulate solution or suspension, or in a particulate structuresuch as a paucilamelar liposome or ISCOM®. The saponins may also beformulated with excipients such as CARBOPOL® to increase viscosity, ormay be formulated in a dry powder form with a powder excipient such aslactose.

In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 is disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

Additional illustrative adjuvants for use in the pharmaceuticalcompositions of the invention include MONTANIDE® ISA 720 (Seppic,France), SAF™ (Chiron, Calif., United States), ISCOM®s (CSL), MF-59™(Chiron), the SBAS™ series of adjuvants (e.g., SBAS™-2 or SBAS™-4,available from SmithKline Beecham, Rixensart, Belgium), Detox(ENHANZYN®) (Corixa, Hamilton, Mont.), RC-529™ (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720, and in U.S. Pat. No. 6,303,347 the disclosures of which areincorporated herein by reference in their entireties, andpolyoxyethylene ether adjuvants such as those described in WO99/52549A1. Additional illustrative adjuvants for use in thepharmaceutical compositions of the invention include isotucerosol.

Other preferred adjuvants include adjuvant molecules of the generalformula

HO(CH₂CH₂O)_(n)-A-R,  (I)

wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or PhenylC₁₋₅₀ alkyl.

One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₋₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

The polyoxyethylene ether according to the general formula (I) abovemay, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

According to another embodiment of this invention, an immunogeniccomposition described herein is delivered to a host via antigenpresenting cells (APCs), such as dendritic cells, macrophages, B cells,monocytes and other cells that may be engineered to be efficient APCs.Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-tumor effects per seand/or to be immunologically compatible with the receiver (i.e., matchedHLA haplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaïve T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1 BB).

APCs may generally be transfected with a polynucleotide of the invention(or portion or other variant thereof) such that the encoded polypeptide,or an immunogenic portion thereof, is expressed on the cell surface.Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the tumor polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will typically vary depending on the mode ofadministration. Compositions of the present invention may be formulatedfor any appropriate manner of administration, including for example,topical, oral, nasal, mucosal, intravenous, intracranial,intraperitoneal, subcutaneous and intramuscular administration.

Carriers for use within such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation preferably provides a relatively constant level ofactive component release. In other embodiments, however, a more rapidrate of release immediately upon administration may be desired. Theformulation of such compositions is well within the level of ordinaryskill in the art using known techniques. Illustrative carriers useful inthis regard include microparticles of poly(lactide-co-glycolide),polyacrylate, latex, starch, cellulose, dextran and the like. Otherillustrative delayed-release carriers include supramolecular biovectors,which comprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g.,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

In another illustrative embodiment, biodegradable microspheres (e.g.,polylactate polyglycolate) are employed as carriers for the compositionsof this invention. Suitable biodegradable microspheres are disclosed,for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647;5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252.Modified hepatitis B core protein carrier systems such as described inWO/99 40934, and references cited therein, will also be useful for manyapplications. Another illustrative carrier/delivery system employs acarrier comprising particulate-protein complexes, such as thosedescribed in U.S. Pat. No. 5,928,647, which are capable of inducing aclass 1-restricted cytotoxic T lymphocyte responses in a host.

In another illustrative embodiment, calcium phosphate core particles areemployed as carriers, vaccine adjuvants, or as controlled releasematrices for the compositions of this invention. Exemplary calciumphosphate particles are disclosed, for example, in published patentapplication No. WO/0046147.

The pharmaceutical compositions of the invention will often furthercomprise one or more buffers (e.g., neutral buffered saline or phosphatebuffered saline), carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation, is well known in the art,some of which are briefly discussed below for general purposes ofillustration.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (see, for example,Mathiowitz et al., Nature 1997 Mar. 27; 386(6623):410-4; Hwang et al.,Crit. Rev Ther Drug Carrier Syst 1998; 15(3):243-84; U.S. Pat. No.5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451).Tablets, troches, pills, capsules and the like may also contain any of avariety of additional components, for example, a binder, such as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.Of course, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

Typically, these formulations will contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. Alternatively, the active ingredientmay be incorporated into an oral solution such as one containing sodiumborate, glycerin and potassium bicarbonate, or dispersed in adentifrice, or added in a therapeutically-effective amount to acomposition that may include water, binders, abrasives, flavoringagents, foaming agents, and humectants. Alternatively the compositionsmay be fashioned into a tablet or solution form that may be placed underthe tongue or otherwise dissolved in the mouth.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat.No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. Moreover, for human administration, preparationswill of course preferably meet sterility, pyrogenicity, and the generalsafety and purity standards as required by FDA Office of Biologicsstandards.

In another embodiment of the invention, the compositions disclosedherein may be formulated in a neutral or salt form. Illustrativepharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212.Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., J Controlled Release 1998 Mar. 2; 52(1-2):81-7) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are alsowell-known in the pharmaceutical arts. Likewise, illustrativetransmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol 1998 July; 16(7):307-21;Takakura, Nippon Rinsho 1998 March; 56(3):691-5; Chandran et al., IndianJ Exp Biol. 1997 August; 35(8):801-9; Margalit, Crit. Rev Ther DrugCarrier Syst. 1995; 12(2-3):233-61; U.S. Pat. No. 5,567,434; U.S. Pat.No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety).

Liposomes have been used successfully with a number of cell types thatare normally difficult to transfect by other procedures, including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J Biol. Chem. 1990 Sep. 25; 265(27):16337-42; Muller et al., DNACell Biol. 1990 April; 9(3):221-9). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes,various drugs, radiotherapeutic agents, enzymes, viruses, transcriptionfactors, allosteric effectors and the like, into a variety of culturedcell lines and animals. Furthermore, he use of liposomes does not appearto be associated with autoimmune responses or unacceptable toxicityafter systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that aredispersed in an aqueous medium and spontaneously form multilamellarconcentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. 1998 December; 24(12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1 μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit. Rev Ther Drug Carrier Syst. 1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 March;45(2):149-55; Zambaux et al. J Controlled Release. 1998 Jan. 2;50(1-3):31-40; and U.S. Pat. No. 5,145,684.

Therapy of Malignant Diseases

Immunologic approaches to cancer therapy are based on the recognitionthat cancer cells can often evade the body's defenses against aberrantor foreign cells and molecules, and that these defenses might betherapeutically stimulated to regain the lost ground, e.g. pgs. 623-648in Klein, Immunology (Wiley-Interscience, New York, 1982). Numerousrecent observations that various immune effectors can directly orindirectly inhibit growth of tumors has led to renewed interest in thisapproach to cancer therapy, e.g. Jager, et al., Oncology 2001;60(1):1-7; Renner, et al., Ann Hematol 2000 December; 79(12):651-9.

Four-basic cell types whose function has been associated with antitumorcell immunity and the elimination of tumor cells from the body are: i)B-lymphocytes which secrete immunoglobulins into the blood plasma foridentifying and labeling the nonself invader cells; ii) monocytes whichsecrete the complement proteins that are responsible for lysing andprocessing the immunoglobulin-coated target invader cells; iii) naturalkiller lymphocytes having two mechanisms for the destruction of tumorcells, antibody-dependent cellular cytotoxicity and natural killing; andiv) T-lymphocytes possessing antigen-specific receptors and having thecapacity to recognize a tumor cell carrying complementary markermolecules (Schreiber, H., 1989, in Fundamental Immunology (ed). W. E.Paul, pp. 923-955).

Cancer immunotherapy generally focuses on inducing humoral immuneresponses, cellular immune responses, or both. Moreover, it is wellestablished that induction of CD4⁺ T helper cells is necessary in orderto secondarily induce either antibodies or cytotoxic CD8⁺ T cells.Polypeptide antigens that are selective or ideally specific for cancercells, particularly cancer cells associated with WT1 expression, offer apowerful approach for inducing immune responses against cancerassociated with WT1 expression, and are an important aspect of thepresent invention.

In further aspects of the present invention, the compositions andvaccines described herein may be used to inhibit the development ofmalignant diseases (e.g., progressive or metastatic diseases or diseasescharacterized by small tumor burden such as minimal residual disease).In general, such methods may be used to prevent, delay or treat adisease associated with WT1 expression. In other words, therapeuticmethods provided herein may be used to treat an existing WT1-associateddisease, or may be used to prevent or delay the onset of such a diseasein a patient who is free of disease or who is afflicted with a diseasethat is not yet associated with WT1 expression.

As used herein, a disease is “associated with WT1 expression” ifdiseased cells (e.g., tumor cells) at some time during the course of thedisease generate detectably higher levels of a WT1 polypeptide thannormal cells of the same tissue. Association of WT1 expression with amalignant disease does not require that WT1 be present on a tumor. Forexample, overexpression of WT1 may be involved with initiation of atumor, but the protein expression may subsequently be lost.Alternatively, a malignant disease that is not characterized by anincrease in WT1 expression may, at a later time, progress to a diseasethat is characterized by increased WT1 expression. Accordingly, anymalignant disease in which diseased cells formerly expressed, currentlyexpress or are expected to subsequently express increased levels of WT1is considered to be “associated with WT1 expression.”

Immunotherapy may be performed using any of a variety of techniques, inwhich compounds or cells provided herein function to removeWT1-expressing cells from a patient. Such removal may take place as aresult of enhancing or inducing an immune response in a patient specificfor WT1 or a cell expressing WT1. Alternatively, WT1-expressing cellsmay be removed ex vivo (e.g., by treatment of autologous bone marrow,peripheral blood or a fraction of bone marrow or peripheral blood).Fractions of bone marrow or peripheral blood may be obtained using anystandard technique in the art.

Within such methods, pharmaceutical compositions and vaccines may beadministered to a patient. As used herein, a “patient” refers to anywarm-blooded animal, preferably a human. A patient may or may not beafflicted with a malignant disease. Accordingly, the abovepharmaceutical compositions and vaccines may be used to prevent theonset of a disease (i.e., prophylactically) or to treat a patientafflicted with a disease (e.g., to prevent or delay progression and/ormetastasis of an existing disease). A patient afflicted with a diseasemay have a minimal residual disease (e.g., a low tumor burden in aleukemia patient in complete or partial remission or a cancer patientfollowing reduction of the tumor burden after surgery radiotherapyand/or chemotherapy). Such a patient may be immunized to inhibit arelapse (i.e., prevent or delay the relapse, or decrease the severity ofa relapse). Within certain preferred embodiments, the patient isafflicted with a leukemia (e.g., AML, CML, ALL or childhood ALL), amyelodysplastic syndrome (MDS) or a cancer (e.g., gastrointestinal,lung, thyroid or breast cancer or a melanoma), where the cancer orleukemia is WT1 positive (i.e., reacts detectably with an anti-WT1antibody, as provided herein or expresses WT1 mRNA at a level detectableby RT-PCR, as described herein) or suffers from an autoimmune diseasedirected against WT1-expressing cells.

Other diseases associated with WT1 overexpression include kidney cancer(such as renal cell carcinoma, or Wilms tumor), as described in SatohF., et al., Pathol. Int. 50(6):458-71 (2000), and Campbell C. E. et al.,Int. J. Cancer 78(2):182-8 (1998); and mesothelioma, as described inAmin, K. M. et al., Am. J. Pathol. 146(2):344-56 (1995). Harada et al.(Mol. Urol. 3(4):357-364 (1999) describe WT1 gene expression in humantesticular germ-cell tumors. Nonomura et al. Hinyokika Kiyo 45(8):593-7(1999) describe molecular staging of testicular cancer using polymerasechain reaction of the testicular cancer-specific genes. Shimizu et al.,Int. J. Gynecol. Pathol. 19(2):158-63 (2000) describe theimmunohistochemical detection of the Wilms' tumor gene (WT1) inepithelial ovarian tumors.

WT1 overexpression was also described in desmoplastic small round celltumors, by Barnoud, R. et al., Am. J. Surg. Pathol. 24(6):830-6 (2000);and Pathol. Res. Pract. 194(10):693-700 (1998). WT1 overexpression inglioblastoma and other cancer was described by Menssen, H. D. et al., J.Cancer Res. Clin. Oncol. 126(4):226-32 (2000), “Wilms' tumor gene (WT1)expression in lung cancer, colon cancer and glioblastoma cell linescompared to freshly isolated tumor specimens.” Other diseases showingWT1 overexpression include EBV associated diseases, such as Burkitt'slymphoma and nasopharyngeal cancer (Spinsanti P. et al., Leuk. Lymphoma38(5-6):611-9 (2000), “Wilms' tumor gene expression by normal andmalignant human B lymphocytes.”

In Leukemia 14(9):1634-4 (2000), Pan et al., describe in vitro IL-12treatment of peripheral blood mononuclear cells from patients withleukemia or myelodysplastic syndromes, and reported an increase incytotoxicity and reduction in WT1 gene expression. In Leukemia13(6):891-900 (1999), Patmasiriwat et al. reported WT1 and GATALexpression in myelodysplastic syndrome and acute leukemia. In Leukemia13(3):393-9 (1999), Tamaki et al. reported that the Wilms' tumor geneWT1 is a good marker for diagnosis of disease progression ofmyelodysplastic syndromes. Expression of the Wilms' tumor gene WT1 insolid tumors, and its involvement in tumor cell growth, was discussed inrelation to gastric cancer, colon cancer, lung cancer, breast cancercell lines, germ cell tumor cell line, ovarian cancer, the uterinecancer, thyroid cancer cell line, hepatocellular carcinoma, in Oji etal., Jpn. J. Cancer Res. 90(2):194-204 (1999).

The compositions provided herein may be used alone or in combinationwith conventional therapeutic regimens such as surgery, irradiation,chemotherapy and/or bone marrow transplantation (autologous, syngeneic,allogeneic or unrelated). As discussed in greater detail below, bindingagents and T cells as provided herein may be used for purging ofautologous stem cells. Such purging may be beneficial prior to, forexample, bone marrow transplantation or transfusion of blood orcomponents thereof. Binding agents, T cells, antigen presenting cells(APC) and compositions provided herein may further be used for expandingand stimulating (or priming) autologous, allogeneic, syngeneic orunrelated WT1-specific T-cells in vitro and/or in vivo. SuchWT1-specific T cells may be used, for example, within donor lymphocyteinfusions.

Routes and frequency of administration, as well as dosage, will varyfrom individual to individual, and may be readily established usingstandard techniques. In general, the pharmaceutical compositions andvaccines may be administered by injection (e.g., intracutaneous,intramuscular, intravenous or subcutaneous), intranasally (e.g., byaspiration) or orally. In some tumors, pharmaceutical compositions orvaccines may be administered locally (by, for example, rectocoloscopy,gastroscopy, videoendoscopy, angiography or other methods known in theart). Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-tumor immune responsethat is at least 10-50% above the basal (i.e., untreated) level. Suchresponse can be monitored by measuring the anti-tumor antibodies in apatient or by vaccine-dependent generation of cytolytic effector cellscapable of killing the patient's tumor cells in vitro. Such vaccinesshould also be capable of causing an immune response that leads to animproved clinical outcome (e.g., more frequent complete or partialremissions, or longer disease-free and/or overall survival) invaccinated patients as compared to non-vaccinated patients. In general,for pharmaceutical compositions and vaccines comprising one or morepolypeptides, the amount of each polypeptide present in a dose rangesfrom about 100 μg to 5 mg. Suitable dose sizes will vary with the sizeof the patient, but will typically range from about 0.1 mL to about 5mL.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent complete or partialremissions, or longer disease-free and/or overall survival) in treatedpatients as compared to non-treated patients. Increases in preexistingimmune responses to WT1 generally correlate with an improved clinicaloutcome. Such immune responses may generally be evaluated using standardproliferation, cytotoxicity or cytokine assays, which may be performedusing samples obtained from a patient before and after treatment.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against tumors with the administration ofimmune response-modifying agents (such as polypeptides andpolynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as effector cells or antibodies) that candirectly or indirectly mediate antitumor effects and does notnecessarily depend on an intact host immune system. Examples of effectorcells include T cells as discussed above, T lymphocytes (such as CD8⁺cytotoxic T lymphocytes and CD4⁺ T-helper tumor-infiltratinglymphocytes), killer cells (such as Natural Killer cells andlymphokine-activated killer cells), B cells and antigen-presenting cells(such as dendritic cells and macrophages) expressing a polypeptideprovided herein. T cell receptors and antibody receptors specific forthe polypeptides recited herein may be cloned, expressed and transferredinto other vectors or effector cells for adoptive immunotherapy. Thepolypeptides provided herein may also be used to generate antibodies oranti-idiotypic antibodies (as described above and in U.S. Pat. No.4,918,164) for passive immunotherapy.

Monoclonal antibodies may be labeled with any of a variety of labels fordesired selective usages in detection, diagnostic assays or therapeuticapplications (as described in U.S. Pat. Nos. 6,090,365; 6,015,542;5,843,398; 5,595,721; and 4,708,930, hereby incorporated by reference intheir entirety as if each was incorporated individually). In each case,the binding of the labelled monoclonal antibody to the determinant siteof the antigen will signal detection or delivery of a particulartherapeutic agent to the antigenic determinant on the non-normal cell. Afurther object of this invention is to provide the specific monoclonalantibody suitably labelled for achieving such desired selective usagesthereof.

Effector cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

Alternatively, a vector expressing a polypeptide recited herein may beintroduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal or intratumor administration.

Within further aspects, methods for inhibiting the development of amalignant disease associated with WT1 expression involve theadministration of autologous T cells that have been activated inresponse to a WT1 polypeptide or WT1-expressing APC, as described above.Such T cells may be CD4⁺ and/or CD8⁺, and may be proliferated asdescribed above. The T cells may be administered to the individual in anamount effective to inhibit the development of a malignant disease.Typically, about 1×10⁹ to 1×10¹¹ T cells/M² are administeredintravenously, intracavitary or in the bed of a resected tumor. It willbe evident to those skilled in the art that the number of cells and thefrequency of administration will be dependent upon the response of thepatient.

Within certain embodiments, T cells may be stimulated prior to anautologous bone marrow transplantation. Such stimulation may take placein vivo or in vitro. For in vitro stimulation, bone marrow and/orperipheral blood (or a fraction of bone marrow or peripheral blood)obtained from a patient may be contacted with a WT1 polypeptide, apolynucleotide encoding a WT1 polypeptide and/or an APC that expresses aWT1 polypeptide under conditions and for a time sufficient to permit thestimulation of T cells as described above. Bone marrow, peripheral bloodstem cells and/or WT1-specific T cells may then be administered to apatient using standard techniques.

Within related embodiments, T cells of a related or unrelated donor maybe stimulated prior to a syngeneic or allogeneic (related or unrelated)bone marrow transplantation. Such stimulation may take place in vivo orin vitro. For in vitro stimulation, bone marrow and/or peripheral blood(or a fraction of bone marrow or peripheral blood) obtained from arelated or unrelated donor may be contacted with a WT1 polypeptide, WT1polynucleotide and/or APC that expresses a WT1 polypeptide underconditions and for a time sufficient to permit the stimulation of Tcells as described above. Bone marrow, peripheral blood stem cellsand/or WT1-specific T cells may then be administered to a patient usingstandard techniques.

Within other embodiments, WT1-specific T cells as described herein maybe used to remove cells expressing WT1 from autologous bone marrow,peripheral blood or a fraction of bone marrow or peripheral blood (e.g.,CD34⁺ enriched peripheral blood (PB) prior to administration to apatient). Such methods may be performed by contacting bone marrow or PBwith such T cells under conditions and for a time sufficient to permitthe reduction of WT1 expressing cells to less than 10%, preferably lessthan 5% and more preferably less than 1%, of the total number of myeloidor lymphatic cells in the bone marrow or peripheral blood. The extent towhich such cells have been removed may be readily determined by standardmethods such as, for example, qualitative and quantitative PCR analysis,morphology, immunohistochemistry and FACS analysis. Bone marrow or PB(or a fraction thereof) may then be administered to a patient usingstandard techniques.

Cancer Detection and Diagnostic Compositions, Methods and Kits

In general, a cancer associated with WT1 expression may be detected in apatient based on the presence of one or more WT1 proteins and/orpolynucleotides encoding such proteins in a biological sample (forexample, blood, sera, sputum urine and/or tumor biopsies) obtained fromthe patient. In other words, such WT1 proteins may be used as markers toindicate the presence or absence of a cancer. The binding agentsprovided herein generally permit detection of the level of antigen thatbinds to the agent in the biological sample.

Polynucleotide primers and probes may be used to detect the level ofmRNA encoding a WT1 protein, which is also indicative of the presence orabsence of a cancer. In general, a WT1 sequence should be present at alevel that is at least two-fold, preferably three-fold, and morepreferably five-fold or higher in tumor tissue than in normal tissue ofthe same type from which the tumor arose. Expression levels of WT1 intissue types different from that in which the tumor arose are irrelevantin certain diagnostic embodiments since the presence of tumor cells canbe confirmed by observation of predetermined differential expressionlevels, e.g., 2-fold, 5-fold, etc, in tumor tissue to expression levelsin normal tissue of the same type.

Other differential expression patterns can be utilized advantageouslyfor diagnostic purposes. For example, in one aspect of the invention,overexpression of WT1 sequence in tumor tissue and normal tissue of thesame type, but not in other normal tissue types, e.g. PBMCs, can beexploited diagnostically. In this case, the presence of metastatic tumorcells, for example in a sample taken from the circulation or some othertissue site different from that in which the tumor arose, can beidentified and/or confirmed by detecting expression of the tumorsequence in the sample, for example using RT-PCR analysis. In manyinstances, it will be desired to enrich for tumor cells in the sample ofinterest, e.g., PBMCs, using cell capture or other like techniques.

There are a variety of assay formats known to those of ordinary skill inthe art for using a binding agent to detect WT1 polypeptide markers in asample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In general, the presence or absenceof a cancer associated with WT1 in a patient may be determined by (a)contacting a biological sample obtained from a patient with a bindingagent; (b) detecting in the sample a level of WT1 polypeptide that bindsto the binding agent; and (c) comparing the level of WT1 polypeptidewith a predetermined cut-off value.

In a preferred embodiment, the assay involves the use of binding agentimmobilized on a solid support to bind to and remove the WT1 polypeptidefrom the remainder of the sample. The bound WT1 polypeptide may then bedetected using a detection reagent that contains a reporter group andspecifically binds to the binding agent/WT1 polypeptide complex. Suchdetection reagents may comprise, for example, a binding agent thatspecifically binds to a WT1 polypeptide or an antibody or other agentthat specifically binds to the binding agent, such as ananti-immunoglobulin, protein G, protein A or a lectin. Alternatively, acompetitive assay may be utilized, in which a WT1 polypeptide is labeledwith a reporter group and allowed to bind to the immobilized bindingagent after incubation of the binding agent with the sample. The extentto which components of the sample inhibit the binding of the labeled WT1polypeptide to the binding agent is indicative of the reactivity of thesample with the immobilized binding agent. Suitable polypeptides for usewithin such assays include full length WT1 proteins and polypeptideportions thereof to which the binding agent binds, as described above.

The solid support may be any material known to those of ordinary skillin the art to which the WT1 protein may be attached. For example, thesolid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the binding agent. For example, the bindingagent may be covalently attached to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on the bindingpartner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991,at A12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. Thisassay may be performed by first contacting an antibody that has beenimmobilized on a solid support, commonly the well of a microtiter plate,with the sample, such that WT1 polypeptides within the sample areallowed to bind to the immobilized antibody. Unbound sample is thenremoved from the immobilized polypeptide-antibody complexes and adetection reagent (preferably a second antibody capable of binding to adifferent site on the polypeptide) containing a reporter group is added.The amount of detection reagent that remains bound to the solid supportis then determined using a method appropriate for the specific reportergroup.

More specifically, once the antibody is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or TWEEN® 20(Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is thenincubated with the sample, and polypeptide is allowed to bind to theantibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of WT1 polypeptide within asample obtained from an individual with a cancer associated with WT1least about 95% of that achieved at equilibrium between bound andunbound polypeptide. Those of ordinary skill in the art will recognizethat the time necessary to achieve equilibrium may be readily determinedby assaying the level of binding that occurs over a period of time. Atroom temperature, an incubation time of about 30 minutes is generallysufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% TWEEN® 20. The secondantibody, which contains a reporter group, may then be added to thesolid support. Preferred reporter groups include those groups recitedabove.

The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of a cancer associated with WT1expression the signal detected from the reporter group that remainsbound to the solid support is generally compared to a signal thatcorresponds to a predetermined cut-off value. In one preferredembodiment, the cut-off value for the detection of a cancer associatedwith WT1 is the average mean signal obtained when the immobilizedantibody is incubated with samples from patients without the cancer. Ingeneral, a sample generating a signal that is three standard deviationsabove the predetermined cut-off value is considered positive for thecancer. In an alternate preferred embodiment, the cut-off value isdetermined using a Receiver Operator Curve, according to the method ofSackett et al., Clinical Epidemiology: A Basic Science for ClinicalMedicine, Little Brown and Co., 1985, p. 106-7. Briefly, in thisembodiment, the cut-off value may be determined from a plot of pairs oftrue positive rates (i.e., sensitivity) and false positive rates(100%-specificity) that correspond to each possible cut-off value forthe diagnostic test result. The cut-off value on the plot that is theclosest to the upper left-hand corner (i.e., the value that encloses thelargest area) is the most accurate cut-off value, and a samplegenerating a signal that is higher than the cut-off value determined bythis method may be considered positive. Alternatively, the cut-off valuemay be shifted to the left along the plot, to minimize the falsepositive rate, or to the right, to minimize the false negative rate. Ingeneral, a sample generating a signal that is higher than the cut-offvalue determined by this method is considered positive for a cancer.

In a related embodiment, the assay is performed in a flow-through orstrip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of a cancer. Typically, the concentration of second bindingagent at that site generates a pattern, such as a line, that can be readvisually. The absence of such a pattern indicates a negative result. Ingeneral, the amount of binding agent immobilized on the membrane isselected to generate a visually discernible pattern when the biologicalsample contains a level of polypeptide that would be sufficient togenerate a positive signal in the two-antibody sandwich assay, in theformat discussed above. Preferred binding agents for use in such assaysare antibodies and antigen-binding fragments thereof. Preferably, theamount of antibody immobilized on the membrane ranges from about 25 ngto about 1 μg, and more preferably from about 50 ng to about 500 ng.Such tests can typically be performed with a very small amount ofbiological sample.

Of course, numerous other assay protocols exist that are suitable foruse with the WT1 proteins or binding agents of the present invention.The above descriptions are intended to be exemplary only. For example,it will be apparent to those of ordinary skill in the art that the aboveprotocols may be readily modified to use tumor polypeptides to detectantibodies that bind to such polypeptides in a biological sample. Thedetection of such WT1-specific antibodies may correlate with thepresence of a cancer associated with WT1 expression.

A cancer associated with WT1 expression may also, or alternatively, bedetected based on the presence of T cells that specifically react with atumor protein in a biological sample. Within certain methods, abiological sample comprising CD4⁺ and/or CD8⁺ T cells isolated from apatient is incubated with a WT1 polypeptide, a polynucleotide encodingsuch a polypeptide and/or an APC that expresses at least an immunogenicportion of such a polypeptide, and the presence or absence of specificactivation of the T cells is detected. Suitable biological samplesinclude, but are not limited to, isolated T cells. For example, T cellsmay be isolated from a patient by routine techniques (such as byFicoll/Hypaque density gradient centrifugation of peripheral bloodlymphocytes). T cells may be incubated in vitro for 2-9 days (typically4 days) at 37 C with polypeptide (e.g., 5-25 g/ml). It may be desirableto incubate another aliquot of a T cell sample in the absence of WT1polypeptide to serve as a control. For CD4⁺ T cells, activation ispreferably detected by evaluating proliferation of the T cells. For CD8⁺T cells, activation is preferably detected by evaluating cytolyticactivity. A level of proliferation that is at least two fold greaterand/or a level of cytolytic activity that is at least 20% greater thanin disease-free patients indicates the presence of a cancer associatedwith WT1 expression in the patient.

As noted above, a cancer may also, or alternatively, be detected basedon the level of mRNA encoding a WT1 protein in a biological sample. Forexample, at least two oligonucleotide primers may be employed in apolymerase chain reaction (PCR) based assay to amplify a portion of aWT1 cDNA derived from a biological sample, wherein at least one of theoligonucleotide primers is specific for (i.e., hybridizes to) apolynucleotide encoding the WT1 protein. The amplified cDNA is thenseparated and detected using techniques well known in the art, such asgel electrophoresis.

Similarly, oligonucleotide probes that specifically hybridize to apolynucleotide encoding a WT1 protein may be used in a hybridizationassay to detect the presence of polynucleotide encoding the WT1 proteinin a biological sample.

To permit hybridization under assay conditions, oligonucleotide primersand probes should comprise an oligonucleotide sequence that has at leastabout 60%, preferably at least about 75% and more preferably at leastabout 90%, identity to a portion of a polynucleotide encoding a WT1protein of the invention that is at least 10 nucleotides, and preferablyat least 20 nucleotides, in length. Preferably, oligonucleotide primersand/or probes hybridize to a polynucleotide encoding a polypeptidedescribed herein under moderately stringent conditions, as definedabove. Oligonucleotide primers and/or probes which may be usefullyemployed in the diagnostic methods described herein preferably are atleast 10-40 nucleotides in length. In a preferred embodiment, theoligonucleotide primers comprise at least 10 contiguous nucleotides,more preferably at least 15 contiguous nucleotides, of a DNA moleculehaving a sequence as disclosed herein. Techniques for both PCR basedassays and hybridization assays are well known in the art (see, forexample, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263,1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989).

One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample, such as biopsy tissue, and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not afflicted with a cancer. The amplification reaction may beperformed on several dilutions of cDNA spanning two orders of magnitude.A two-fold or greater increase in expression in several dilutions of thetest patient sample as compared to the same dilutions of thenon-cancerous sample is typically considered positive.

In another aspect of the present invention, cell capture technologiesmay be used in conjunction, with, for example, real-time PCR to providea more sensitive tool for detection of metastatic cells expressing WT1antigens. Detection of WT1-associated cancer cells in biologicalsamples, e.g., bone marrow samples, peripheral blood, and small needleaspiration samples is desirable for diagnosis and prognosis in patientswith cancer associated with WT1 expression.

Immunomagnetic beads coated with specific monoclonal antibodies tosurface cell markers, or tetrameric antibody complexes, may be used tofirst enrich or positively select cancer cells in a sample. Variouscommercially available kits may be used, including DYNABEADS® EpithelialEnrich (Dynal Biotech, Oslo, Norway), STEMSEP® (StemCell Technologies,Inc., Vancouver, BC), and ROSETTESEP® (StemCell Technologies). A skilledartisan will recognize that other methodologies and kits may also beused to enrich or positively select desired cell populations. DYNABEADS®Epithelial Enrich contains magnetic beads coated with mAbs specific fortwo glycoprotein membrane antigens expressed on normal and neoplasticepithelial tissues. The coated beads may be added to a sample and thesample then applied to a magnet, thereby capturing the cells bound tothe beads. The unwanted cells are washed away and the magneticallyisolated cells eluted from the beads and used in further analyses.

ROSETTESEP® can be used to enrich cells directly from a blood sample andconsists of a cocktail of tetrameric antibodies that targets a varietyof unwanted cells and cross-links them to glycophorin A on red bloodcells (RBC) present in the sample, forming rosettes. When centrifugedover FICOLL®, targeted cells pellet along with the free RBC. Thecombination of antibodies in the depletion cocktail determines whichcells will be removed and consequently which cells will be recovered.Antibodies that are available include, but are not limited to: CD2, CD3,CD4, CD5, CD8, CD10, CD11b, CD14, CD15, CD16, CD19, CD20, CD24, CD25,CD29, CD33, CD34, CD36, CD38, CD41, CD45, CD45RA, CD45RO, CD56, CD66B,CD66e, HLA-DR, IgE, and TCRαβ.

In another embodiment, the compositions described herein may be used asmarkers for the progression of cancer. In this embodiment, assays asdescribed above for the diagnosis of a cancer associated with WT1expression may be performed over time, and the change in the level ofreactive polypeptide(s) or polynucleotide(s) evaluated. For example, theassays may be performed every 24-72 hours for a period of 6 months to 1year, and thereafter performed as needed. In general, a cancer isprogressing in those patients in whom the level of WT1 polypeptide orpolynucleotide detected increases over time. In contrast, the cancer isnot progressing when the level of reactive polypeptide or polynucleotideeither remains constant or decreases with time.

Certain in vivo diagnostic assays may be performed directly on a tumor.One such assay involves contacting tumor cells with a binding agent. Thebound binding agent may then be detected directly or indirectly via areporter group. Such binding agents may also be used in histologicalapplications. Alternatively, polynucleotide probes may be used withinsuch applications.

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a WT1 protein. Such antibodies orfragments may be provided attached to a support material, as describedabove. One or more additional containers may enclose elements, such asreagents or buffers, to be used in the assay. Such kits may also, oralternatively, contain a detection reagent as described above thatcontains a reporter group suitable for direct or indirect detection ofantibody binding.

Alternatively, a kit may be designed to detect the level of mRNAencoding a WT1 protein in a biological sample. Such kits generallycomprise at least one oligonucleotide probe or primer, as describedabove, that hybridizes to a polynucleotide encoding a WT1 protein. Suchan oligonucleotide may be used, for example, within a PCR orhybridization assay. Additional components that may be present withinsuch kits include a second oligonucleotide and/or a diagnostic reagentor container to facilitate the detection of a polynucleotide encoding aWT1 protein.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Identification of an Immune Response to WT1 inPatients with Hematological Malignancies

This Example illustrates the identification of an existent immuneresponse in patients with a hematological malignancy.

To evaluate the presence of preexisting WT1 specific antibody responsesin patients, sera of patients with acute myelogenous leukemia (AML),acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML) andsevere aplastic anemia were analyzed using Western blot analysis. Serawere tested for the ability to immunoprecipitate WT1 from the humanleukemic cell line K562 (American Type Culture Collection, Manassas,Va.). In each case, immunoprecipitates were separated by gelelectrophoresis, transferred to membrane and probed with the anti WT1antibody WT180 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.).This Western blot analysis identified potential WT1 specific antibodiesin patients with hematological malignancy. A representative Western blotshowing the results for a patient with AML is shown in FIG. 2. A 52 kDprotein in the immunoprecipitate generated using the patient sera wasrecognized by the WT1 specific antibody. The 52 kD protein migrated atthe same size as the positive control.

Additional studies analyzed the sera of patients with AML and CML forthe presence of antibodies to full-length and truncated WT1 proteins.cDNA constructs representing the human WT1/full-length (aa 1-449), theN-terminus (aa 1-249) (WT1/N-terminus) and C-terminus (aa 267-449)(WT1/C-terminus) region were subcloned into modified pET28 vectors. TheWT1/full-length and WT1/N-terminus proteins were expressed as Ra12fusion proteins. Ra12 is the C-terminal fragment of a secretedMycobacterium tuberculosis protein, denoted as MTB32B. (Skeiky et al.,Infect Immun. 67; 3998, 1999). The Ra12-WT1/full-length fusion regionwas cloned 3′ to a histidine-tag in a histidine-tag modified pET28vector. The WT1/N-terminus region was subcloned into a modified pET28vector that has a 5′ histidine-tag followed by the thioredoxin(TRX)-WT1/N-terminus fusion region followed by a 3′ histidine-tag. TheWT1/C-terminus coding region was subcloned into a modified pET28 vectorwithout a fusion partner containing only the 5′ and 3′ histidine-tag,followed by a Thrombin and EK site.

BL21 pLysS E. coli (Stratagene, La Jolla, Calif.) were transformed withthe three WT1 expression constructs, grown overnight and induced withisopropyl-β-D-thiogalactoside (IPTG). WT1 proteins were purified asfollows: Cells were harvested and lysed by incubation in 10 mM Tris, pH8.0 with Complete Protease Inhibitor Tablets (Boehringer MannheimBiochemicals, Indianapolis, Ind.) at 37° C. followed by repeated roundsof sonication. Inclusion bodies were washed twice with 10 mM Tris, pH8.0. Proteins were then purified by metal chelate affinitychromatography over nickel-nitrilotriacetic acid resin (QIAGEN Inc.,Valencia, Calif.; Hochuli et al., Biologically Active Molecules:217,1989) followed by chromatography on a Source Q anion exchange resin(Amersham Pharmacia Biotech, Upsala, Sweden). The identity of the WT1proteins was confirmed by N-terminal sequencing.

Sera from adult patients with de nova AML or CML were studied for thepresence of WT1 specific Ab. Recombinant proteins were adsorbed to TCmicrowell plates (Nunc, Roskilde, Denmark). Plates were washed withPBS/0.5% TWEEN® 20 and blocked with 1% BSA/PBS/0.1% TWEEN® 20. Afterwashing, serum dilutions were added and incubated overnight at 4° C.Plates were washed and Donkey anti-human IgG-HRP secondary antibody wasadded (Jackson-Immunochem, West Grove, Pa.) and incubated for 2 h atroom temperature. Plates were washed, incubated with TMB Peroxidasesubstrate solution (Kirkegaard and Perry Laboratories, Mass.), quenchedwith 1N H₂SO₄, and immediately read (Cyto-Fluor 2350; Millipore,Bedford, Mass.).

For the serological survey, human sera were tested by ELISA over a rangeof serial dilutions from 1:50 to 1:20,000. A positive reaction wasdefined as an OD value of a 1:500 diluted serum that exceeded the meanOD value of sera from normal donors (n=96) by three (WT1/full-length,WT1C-terminus) standard deviations. Due to a higher background in normaldonors to the WT1/N-terminus protein a positive reaction toWT1/N-terminus was defined as an OD value of 1:500 diluted serum thatexceeded the mean OD value of sera from normal donors by four standarddeviations. To verify that the patient Ab response was directed againstWT1 and not to the Ra12 or TRX fusion part of the protein or possible E.coli contaminant proteins, controls included the Ra12 and TRX proteinalone purified in a similar manner. Samples that showed reactivityagainst the Ra12 and/or TRX proteins were excluded from the analysis.

To evaluate for the presence of immunity to WT1, Ab to recombinantfull-length and truncated WT1 proteins in the sera of normal individualsand patients with leukemia were determined. Antibody reactivity wasanalyzed by ELISA reactivity to WT1/full-length protein, WT1/N-terminusprotein and WT1/C-terminus protein.

Only 2 of 96 normal donors had serum antibodies reactive withWT1/full-length protein (FIG. 18). One of those individuals had antibodyto WT1/N-terminus protein and one had antibody to WT1/C-terminusprotein. In contrast, 16 of 63 patients (25%) with AML had serumantibodies reactive with WT1/full-length protein. By marked contrast,only 2 of 63 patients (3%) had reactivity to WT1/C-terminus protein.Fifteen of 81 patients (19%) with CML had serum antibodies reactive withWT1/full-length protein and 12 of 81 patients (15%) had serum antibodiesreactive with WT1/N-terminus. Only 3 of 81 patients (3%) had reactivityto WT1/C-terminus protein. (FIGS. 16 and 17.)

These data demonstrate that Ab responses to WT1 are detectable in somepatients with AML and CML. The greater incidence of antibody in leukemiapatients provides strong evidence that immunization to the WT1 proteinoccurred as a result of patients bearing malignancy that expresses or atsome time expressed WT1. Without being limited to a specific theory, itis believed that the observed antibody responses to WT1 most probablyresult from patients becoming immune to WT1 on their own leukemia cellsand provide direct evidence that WT1 can be immunogenic despite being a“self” protein.

The presence of antibody to WT1 strongly implies that concurrent helperT cell responses are also present in the same patients. WT1 is aninternal protein. Thus, CTL responses are likely to be the mosteffective in terms of leukemia therapy and the most toxic arm ofimmunity. Thus, these data provide evidence that therapeutic vaccinesdirected against WT1 will be able to elicit an immune response to WT1.

The majority of the antibodies detected were reactive with epitopeswithin the N-terminus while only a small subgroup of patients showed aweak antibody response to the C-terminus. This is consistent withobservations in the animal model, where immunization with peptidesderived from the N-terminus elicited antibody, helper T cell and CTLresponses, whereas none of the peptides tested from the C-terminuselicited antibody or T cell responses (Gaiger et al., Blood 96:1334,2000).

Example 2 Induction of Antibodies to WT1 in Mice Immunized with CellLines Expressing WT1

This Example illustrates the use of cells expressing WT1 to induce a WT1specific antibody response in vivo.

Detection of existent antibodies to WT1 in patients with leukemiastrongly implied that it is possible to immunize to WT1 protein toelicit immunity to WT1. To test whether immunity to WT1 can be generatedby vaccination, mice were injected with TRAMP-C, a WT1 positive tumorcell line of B6 origin. Briefly, male B6 mice were immunized with 5×10⁶TRAMP-C cells subcutaneously and boosted twice with 5×10⁶ cells at threeweek intervals. Three weeks after the final immunization, sera wereobtained and single cell suspensions of spleens were prepared in RPMI1640 medium (GIBCO®) with 25CM β-2-mercaptoethanol, 200 units ofpenicillin per ml, 10 mM L-glutamine, and 10% fetal bovine serum.

Following immunization to TRAMP-C, a WT1 specific antibody response inthe immunized animals was detectable. A representative Western blot isshown in FIG. 3. These results show that immunization to WT1 protein canelicit an immune response to WT1 protein.

Example 3 Induction of Th and Antibody Responses in Mice Immunized withWT1 Peptides

This Example illustrates the ability of immunization with WT1 peptidesto elicit an immune response specific for WT1.

Peptides suitable for eliciting Ab and proliferative T cell responseswere identified according to the Tsites program (Rothbard and Taylor,EMBO J. 7:93-100, 1988; Deavin et al., Mol. Immunol. 33:145-155, 1996),which searches for peptide motifs that have the potential to elicit Thresponses. Peptides shown in Table I were synthesized and sequenced.

TABLE I WT1 Peptides Peptide Sequence Comments Mouse: p6-22RDLNALLPAVSSLGGGG 1 mismatch (SEQ ID NO: 13) relative to human WT1sequence Human: p6-22 RDLNALLPAVPSLGGGG (SEQ ID NO: 1) Human/mouse:PSQASSGQARMFPNAPYLPSCLE p117-139 (SEQ ID NOs: 2 and 3) Mouse: p244-GATLKGMAAGSSSSVKWTE 1 mismatch 262 (SEQ ID NO: 14) relative to human WT1sequence Human: p244- GATLKGVAAGSSSSVKWTE 262 (SEQ ID NO: 4)Human/mouse: RIHTHGVFRGIQDVR p287-301 (SEQ ID NOs: 15 and 16) Mouse:p299- VRRVSGVAPTLVRS 1 mismatch 313 (SEQ ID NO: 17) relative to humanWT1 sequence Human/mouse: CQKKFARSDELVRHH p421-435 (SEQ ID NOs: 19 and20)

For immunization, peptides were grouped as follows:

-   -   Group A: p6-22 human: 10.9 mg in 1 ml (10 μl=100 μg)        -   p117-139 human/mouse: 7.6 mg in 1 ml (14 μl=100 μg)        -   p244-262 human: 4.6 mg in 1 ml (22 μl=100 μg)    -   Group B: p287-301 human/mouse: 7.2 mg in 1 ml (14 μl=100 μg)        -   mouse p299-313: 6.6.mg in 1 ml (15 μl=100 μg)        -   p421-435 human/mouse: 3.3 mg in 1 ml (30 μl=100 μg)    -   Control: (FBL peptide 100 μg)+CFA/IFA    -   Control: (CD45 peptide 100 μg)+CFA/IFA

Group A contained peptides present within the amino terminus portion ofWT1 (exon 1) and Group B contained peptides present within the carboxyterminus, which contains a four zinc finger region with sequencehomology to other DNA-binding proteins. Within group B, p287-301 andp299-313 were derived from exon 7, zinc finger 1, and p421-435 wasderived from exon 10, zinc finger IV.

B6 mice were immunized with a group of WT1 peptides or with a controlpeptide. Peptides were dissolved in 1 ml sterile water for injection,and B6 mice were immunized 3 times at time intervals of three weeks.Adjuvants used were CFA/IFA, GM-CSF, and MONTANIDE®. The presence ofantibodies specific for WT1 was then determined as described in Examples1 and 2, and proliferative T cell responses were evaluated using astandard thymidine incorporation assay, in which cells were cultured inthe presence of antigen and proliferation was evaluated by measuringincorporated radioactivity (Chen et al., Cancer Res. 54:1065-1070,1994). In particular, lymphocytes were cultured in 96-well plates at2×10⁵ cells per well with 4×10⁵ irradiated (3000 rads) syngeneic spleencells and the designated peptide.

Immunization of mice with the group of peptides designated as Group Aelicited an antibody response to WT1 (FIG. 4). No antibodies weredetected following immunization to Vaccine B, which is consistent with alack of helper T cell response from immunization with Vaccine B.P117-139 elicited proliferative T cell responses (FIGS. 5A-5C). Thestimulation indices (SI) varied between 8 and 72. Other peptides (P6-22and P299-313) also were shown to elicit proliferative T cell responses.Immunization with P6-22 resulted in a stimulation index (SI) of 2.3 andimmunization with P299-313 resulted in a Si of 3.3. Positive controlsincluded ConA stimulated T cells, as well as T cells stimulated withknown antigens, such as CD45 and FBL, and allogeneic T cell lines(DeBruijn et al., Eur. J. Immunol. 21:2963-2970, 1991).

FIGS. 6A and 6B show the proliferative response observed for each of thethree peptides within vaccine A (FIG. 6A) and vaccine B (FIG. 6B).Vaccine A elicited proliferative T cell responses to the immunizingpeptides p6-22 and p117-139, with stimulation indices (SI) varyingbetween 3 and 8 (bulk lines). No proliferative response to p244-262 wasdetected (FIG. 6A).

Subsequent in vitro stimulations were carried out as single peptidestimulations using only p6-22 and p117-139. Stimulation of the Vaccine Aspecific T cell line with p117-139 resulted in proliferation to p117-139with no response to p6-22 (FIG. 7A). Clones derived from the line werespecific for p117-139 (FIG. 7B). By contrast, stimulation of the VaccineA specific T cell line with p6-22 resulted in proliferation to p6-22with no response to p117-139 (FIG. 7C). Clones derived from the linewere specific for p6-22 (FIG. 7D).

These results show that vaccination with WT1 peptides can elicitantibody responses to WT1 protein and proliferative T cell responses tothe immunizing peptides.

Example 4 Induction of CTL Responses in Mice Immunized with WT1 Peptides

This Example illustrates the ability of WT1 peptides to elicit CTLimmunity.

Peptides (9-mers) with motifs appropriate for binding to class I MHCwere identified using a BIMAS HLA peptide binding prediction analysis(Parker et al., J. Immunol. 152:163, 1994). Peptides identified withinsuch analyses are shown in Tables II-XLIV. In each of these tables, thescore reflects the theoretical binding affinity (half-time ofdissociation) of the peptide to the MHC molecule indicated. Alsoindicated in the tables are the defined peptide binding motifs asdescribed by Rammensee, et al., Immunogenetics 41:178-228, 1995,incorporated herein in its entirety. The peptide binding motif forHLA-B14 is described in DiBrino et al., J. Biol. Chem. 269:23426-23434,1994, also incorporated herein it its entirety. Peptide positions areabbreviated as P1, P2, P3, P4, P5, P6, P7, P8, and P9.

Peptides identified using the Tsites program (Rothbard and Taylor, EMBOJ. 7:93-100, 1988; Deavin et al., Mol. Immunol. 33:145-155, 1996), whichsearches for peptide motifs that have the potential to elicit Thresponses are further shown in FIGS. 8A and 8B, and Table XLV.

TABLE II Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA A1 (HLA-A1 peptide bindingmotif anchor residues are D or E at position 3 (P3); Y at P9; withauxiliary anchors being P at P4 and L at P7) Score (Estimate of HalfTime of Disassociation of Start Subsequence a Molecule Containing RankPosition Residue Listing This Subsequence) 1 137 CLESQPAIR 18.000 (SEQID NO: 47) 2 80 GAEPHEEQC 9.000 (SEQ ID NO: 87) 3 40 FAPPGASAY 5.000(SEQ ID NO: 74) 4 354 QCDFKDCER 5.000 (SEQ ID NO: 162) 5 2 GSDVRDLNA3.750 (SEQ ID NO: 101) 6 152 VTFDGTPSY 2.500 (SEQ ID NO: 244) 7 260WTEGQSNHS 2.250 (SEQ ID NO: 247) 8 409 TSEKPFSCR 1.350 (SEQ ID NO: 232)9 73 KQEPSWGGA 1.350 (SEQ ID NO: 125) 10 386 KTCQRKFSR 1.250 (SEQ ID NO:128) 11 37 VLDFAPPGA 1.000 (SEQ ID NO: 241) 12 325 CAYPGCNKR 1.000 (SEQID NO: 44) 13 232 QLECMTWNQ 0.900 (SEQ ID NO: 167) 14 272 ESDNHTTPI0.750 (SEQ ID NO: 71) 15 366 RSDQLKRHQ 0.750 (SEQ ID NO: 193) 16 222SSDNLYQMT 0.750 (SEQ ID NO: 217) 17 427 RSDELVRHH 0.750 (SEQ ID NO: 191)18 394 RSDHLKTHT 0.750 (SEQ ID NO: 192) 19 317 TSEKRPFMC 0.675 (SEQ IDNO: 233) 20 213 QALLLRTPY 0.500 (SEQ ID NO: 160)

TABLE III Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA A 0201 (HLA-A0201 peptidebinding motif anchor residues are L or M at P2; V or L at P9; with anauxiliary anchor being V at P6) Score (Estimate of Half Time ofDisassociation of Start Subsequence a Molecule Containing Rank PositionResidue Listing This Subsequence) 1 126 RMFPNAPYL 313.968 (SEQ ID NO:185) 2 187 SLGEQQYSV 285.163 (SEQ ID NO: 214) 3 10 ALLPAVPSL 181.794(SEQ ID NO: 34) 4 242 NLGATLKGV 159.970 (SEQ ID NO: 146) 5 225 NLYQMTSQL68.360 (SEQ ID NO: 147) 6 292 GVFRGIQDV 51.790 (SEQ ID NO: 103) 7 191QQYSVPPPV 22.566 (SEQ ID NO: 171) 8 280 ILCGAQYRI 17.736 (SEQ ID NO:116) 9 235 CMTWNQMNL 15.428 (SEQ ID NO: 49) 10 441 NMTKLQLAL 15.428 (SEQID NO: 149) 11 7 DLNALLPAV 11.998 (SEQ ID NO: 58) 12 227 YQMTSQLEC 8.573(SEQ ID NO: 251) 13 239 NQMNLGATL 8.014 (SEQ ID NO: 151) 14 309TLVRSASET 7.452 (SEQ ID NO: 226) 15 408 KTSEKPFSC 5.743 (SEQ ID NO: 129)16 340 LQMHSRKHT 4.752 (SEQ ID NO: 139) 17 228 QMTSQLECM 4.044 (SEQ IDNO: 169) 18 93 TVHFSGQFT 3.586 (SEQ ID NO: 235) 19 37 VLDFAPPGA 3.378(SEQ ID NO: 241) 20 86 EQCLSAFTV 3.068 (SEQ ID NO: 69)

TABLE IV Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA A 0205 (HLA-A0205 peptidebinding motif anchor residues are L at P9 with auxiliary anchors beingV, L, I, M at P2 and I, V, L, A, at P6) Score (Estimate of Half Time ofDisassociation of Start Subsequence a Molecule Containing Rank PositionResidue Listing This Subsequence) 1 10 ALLPAVPSL 42.000 (SEQ ID NO: 34)2 292 GVFRGIQDV 24.000 (SEQ ID NO: 103) 3 126 RMFPNAPYL 21.000 (SEQ IDNO: 185) 4 225 NLYQMTSQL 21.000 (SEQ ID NO: 147) 5 239 NQMNLGATL 16.800(SEQ ID NO: 151) 6 302 RVPGVAPTL 14.000 (SEQ ID NO: 195) 7 441 NMTKLQLAL7.000 (SEQ ID NO: 149) 8 235 CMTWNQMNL 7.000 (SEQ ID NO: 49) 9 187SLGEQQYSV 6.000 (SEQ ID NO: 214) 10 191 QQYSVPPPV 4.800 (SEQ ID NO: 171)11 340 LQMHSRKHT 4.080 (SEQ ID NO: 139) 12 242 NLGATLKGV 4.000 (SEQ IDNO: 146) 13 227 YQMTSQLEC 3.600 (SEQ ID NO: 251) 14 194 SVPPPVYGC 2.000(SEQ ID NO: 218) 15 93 TVHFSGQFT 2.000 (SEQ ID NO: 235) 16 280 ILCGAQYRI1.700 (SEQ ID NO: 116) 17 98 GQFTGTAGA 1.200 (SEQ ID NO: 99) 18 309TLVRSASET 1.000 (SEQ ID NO: 226) 19 81 AEPHEEQCL 0.980 (SEQ ID NO: 30)20 73 KQEPSWGGA 0.960 (SEQ ID NO: 125)

TABLE V Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA A24 (HLA-A24 peptide bindingmotif anchor residues are Y at P2; I, L, or F at P9; with auxiliaryanchors I or V at P5 and F at P6) Score (Estimate of Half Time ofDisassociation of Start Subsequence a Molecule Containing Rank PositionResidue Listing This Subsequence) 1 302 RVPGVAPTL 16.800 (SEQ ID NO:195) 2 218 RTPYSSDNL 12.000 (SEQ ID NO: 194) 3 356 DFKDCERRF 12.000 (SEQID NO: 55) 4 126 RMFPNAPYL 9.600 (SEQ ID NO: 185) 5 326 AYPGCNKRY 7.500(SEQ ID NO: 42) 6 270 GYESDNHT 7.500 (SEQ ID NO: 106) T 7 239 NQMNLGATL7.200 (SEQ ID NO: 151) 8 10 ALLPAVPSL 7.200 (SEQ ID NO: 34) 9 130NAPYLPSCL 7.200 (SEQ ID NO: 144) 10 329 GCNKRYFKL 6.600 (SEQ ID NO: 90)11 417 RWPSCQKKF 6.600 (SEQ ID NO: 196) 12 47 AYGSLGGPA 6.000 (SEQ IDNO: 41) 13 180 DPMGQQGSL 6.000 (SEQ ID NO: 59) 14 4 DVRDLNALL 5.760 (SEQID NO: 62) 15 285 QYRIHTHGV 5.000 (SEQ ID NO: 175) 16 192 QYSVPPPVY5.000 (SEQ ID NO: 176) 17 207 DSCTGSQAL 4.800 (SEQ ID NO: 61) 18 441NMTKLQLAL 4.800 (SEQ ID NO: 149) 19 225 NLYQMTSQL 4.000 (SEQ ID NO: 147)20 235 CMTWNQMNL 4.000 (SEQ ID NO: 49)

TABLE VI Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA A3 (HLA-A3 peptide bindingmotif anchor residues are L, V, or M at P2; K, Y, or F at P9; auxiliaryanchors are F or Y at P3, I, M, F, V, or L at P6; I, L, M, F at P7)Score (Estimate of Half Time of Disassociation of Start Subsequence aMolecule Containing Rank Position Residue Listing This Subsequence) 1436 NMHQRNMTK 40.000 (SEQ ID NO: 148) 2 240 QMNLGATLK 20.000 (SEQ ID NO:168) 3 88 CLSAFTVHF 6.000 (SEQ ID NO: 48) 4 126 RMFPNAPYL 4.500 (SEQ IDNO: 185) 5 169 AQFPNHSFK 4.500 (SEQ ID NO: 36) 6 10 ALLPAVPSL 4.050 (SEQID NO: 34) 7 137 CLESQPAIR 4.000 (SEQ ID NO: 47) 8 225 NLYQMTSQL 3.000(SEQ ID NO: 147) 9 32 AQWAPVLDF 2.700 (SEQ ID NO: 37) 10 280 ILCGAQYRI2.700 (SEQ ID NO: 116) 11 386 KTCQRKFSR 1.800 (SEQ ID NO: 128) 12 235CMTWNQMNL 1.200 (SEQ ID NO: 49) 13 441 NMTKLQLAL 1.200 (SEQ ID NO: 149)14 152 VTFDGTPSY 1.000 (SEQ ID NO: 244) 15 187 SLGEQQYSV 0.900 (SEQ IDNO: 214) 16 383 FQCKTCQRK 0.600 (SEQ ID NO: 80) 17 292 GVFRGIQDV 0.450(SEQ ID NO: 103) 18 194 SVPPPVYGC 0.405 (SEQ ID NO: 218) 19 287RIHTHGVFR 0.400 (SEQ ID NO: 182) 20 263 GQSNHSTGY 0.360 (SEQ ID NO: 100)

TABLE VII Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA A68.1 (HLA-A68.1 peptidebinding motif anchor residues are V or T at P2; R or K at P9 Score(Estimate of Half Time of Disassociation of Start Subsequence a MoleculeContaining Rank Position Residue Listing This Subsequence) 1 100FTGTAGACR 100.000 (SEQ ID NO: 84) 2 386 KTCQRKFSR 50.000 (SEQ ID NO:128) 3 368 DQLKRHQRR 30.000 (SEQ ID NO: 60) 4 312 RSASETSEK 18.000 (SEQID NO: 190) 5 337 LSHLQMHSR 15.000 (SEQ ID NO: 141) 6 364 FSRSDQLKR15.000 (SEQ ID NO: 83) 7 409 TSEKPFSCR 15.000 (SEQ ID NO: 232) 8 299DVRRVPGVA 12.000 (SEQ ID NO: 63) 9 4 DVRDLNALL 12.000 (SEQ ID NO: 62) 10118 SQASSGQAR 10.000 (SEQ ID NO: 216) 11 343 HSRKHTGEK 9.000 (SEQ ID NO:111) 12 169 AQFPNHSFK 9.000 (SEQ ID NO: 36) 13 292 GVFRGIQDV 8.000 (SEQID NO: 103) 14 325 CAYPGCNKR 7.500 (SEQ ID NO: 44) 15 425 FARSDELVR7.500 (SEQ ID NO: 75) 16 354 QCDFKDCER 7.500 (SEQ ID NO: 162) 17 324MCAYPGCNK 6.000 (SEQ ID NO: 142) 18 251 AAGSSSSVK 6.000 (SEQ ID NO: 28)19 379 GVKPFQCKT 6.000 (SEQ ID NO: 104) 20 137 CLESQPAIR 5.000 (SEQ IDNO: 47)

TABLE VIII Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA A 1101 (HLA-A1101 peptidebinding motif anchor residues are K at P9; auxiliary anchor residues areV, I, F, Y at P2, M, L, F, Y, I, A, at P3, L, I, Y, V, or F at P7) Score(Estimate of Half Time of Disassociation of Start Subsequence a MoleculeContaining Rank Position Residue Listing This Subsequence) 1 386KTCQRKFSR 1.800 (SEQ ID NO: 128) 2 169 AQFPNHSFK 1.200 (SEQ ID NO: 36) 3436 NMHQRNMTK 0.800 (SEQ ID NO: 148) 4 391 KFSRSDHLK 0.600 (SEQ ID NO:120) 5 373 HQRRHTGVK 0.600 (SEQ ID NO: 109) 6 383 FQCKTCQRK 0.600 (SEQID NO: 80) 7 363 RFSRSDQLK 0.600 (SEQ ID NO: 178) 8 240 QMNLGATLK 0.400(SEQ ID NO: 168) 9 287 RIHTHGVFR 0.240 (SEQ ID NO: 182) 10 100 FTGTAGACR0.200 (SEQ ID NO: 84) 11 324 MCAYPGCNK 0.200 (SEQ ID NO: 142) 12 251AAGSSSSVK 0.200 (SEQ ID NO: 28) 13 415 SCRWPSCQK 0.200 (SEQ ID NO: 201)14 118 SQASSGQAR 0.120 (SEQ ID NO: 216) 15 292 GVFRGIQDV 0.120 (SEQ IDNO: 103) 16 137 CLESQPAIR 0.080 (SEQ ID NO: 47) 17 425 FARSDELVR 0.080(SEQ ID NO: 75) 18 325 CAYPGCNKR 0.080 (SEQ ID NO: 44) 19 312 RSASETSEK0.060 (SEQ ID NO: 190) 20 65 PPPPHSFIK 0.060 (SEQ ID NO: 156)

TABLE IX Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA A 3101 (HLA-A3101 peptidebinding motif anchor residues are R at P9; auxiliary anchors L, V, Y, orF at P2; F, L, Y, W at P3, L, F, V, I, at P6) Score (Estimate of HalfTime of Dis- association of a Molecule Containing Start Subsequence ThisSub- Rank Position Residue Listing sequence) 1 386 KTCQRKFSR (SEQ ID NO:128) 9.000 2 287 RIHTHGVFR (SEQ ID NO: 182) 6.000 3 137 CLESQPAIR (SEQID NO: 47) 2.000 4 118 SQASSGQAR (SEQ ID NO: 216) 2.000 5 368 DQLKRHQRR(SEQ ID NO: 60) 1.200 6 100 FTGTAGACR (SEQ ID NO: 84) 1.000 7 293VFRGIQDVR (SEQ ID NO: 238) 0.600 8 325 CAYPGCNKR (SEQ ID NO: 44) 0.600 9169 AQFPNHSFK (SEQ ID NO: 36) 0.600 10 279 PILCGAQYR (SEQ ID NO: 155)0.400 11 436 NMHQRNMTK (SEQ ID NO: 148) 0.400 12 425 FARSDELVR (SEQ IDNO: 75) 0.400 13 32 AQWAPVLDF (SEQ ID NO: 37) 0.240 14 240 QMNLGATLK(SEQ ID NO: 168) 0.200 15 354 QCDFKDCER (SEQ ID NO: 162) 0.200 16 373HQRRHTGVK (SEQ ID NO: 109) 0.200 17 383 FQCKTCQRK (SEQ ID NO: 80) 0.20018 313 SASETSEKR (SEQ ID NO: 197) 0.200 19 358 KDCERRFSR (SEQ ID NO:118) 0.180 20 391 KFSRSDHLK (SEQ ID NO: 120) 0.180

TABLE X Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA A 3302 (HLA-A3302 peptidebinding motif anchor residues are R at P9; auxiliary anchors A, I, L, F,Y, or V at P2) Score (Estimate of Half Time of Dis- association of aMolecule Containing Start Subsequence This Sub- Rank Position ResidueListing sequence) 1 337 LSHLQMHSR (SEQ ID NO: 141) 15.000 2 409TSEKPFSCR (SEQ ID NO: 232) 15.000 3 364 FSRSDQLKR (SEQ ID NO: 83) 15.0004 137 CLESQPAIR (SEQ ID NO: 47) 9.000 5 368 DQLKRHQRR (SEQ ID NO: 60)9.000 6 287 RIHTHGVFR (SEQ ID NO: 182) 4.500 7 210 TGSQALLLR (SEQ ID NO:223) 3.000 8 425 FARSDELVR (SEQ ID NO: 75) 3.000 9 313 SASETSEKR (SEQ IDNO: 197) 3.000 10 293 VFRGIQDVR (SEQ ID NO: 238) 3.000 11 354 QCDFKDCER(SEQ ID NO: 162) 3.000 12 100 FTGTAGACR (SEQ ID NO: 84) 3.000 13 118SQASSGQAR (SEQ ID NO: 216) 3.000 14 325 CAYPGCNKR (SEQ ID NO: 44) 3.00015 207 DSCTGSQAL (SEQ ID NO: 61) 1.500 16 139 ESQPAIRNQ (SEQ ID NO: 72)1.500 17 299 DVRRVPGVA (SEQ ID NO: 63) 1.500 18 419 PSCQKKFAR (SEQ IDNO: 159) 1.500 19 272 ESDNHTTPI (SEQ ID NO: 71) 1.500 20 4 DVRDLNALL(SEQ ID NO: 62) 1.500

TABLE XI Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B14 (HLA-B14 peptide bindingmotif anchor residues are R at P2, Y at P3, R at P5, L at P9. Three ofthe 4 anchor residues is sufficient) Score (Estimate of Half Time ofDis- association of a Molecule Containing Start Subsequence This Sub-Rank Position Residue Listing sequence) 1 362 RRFSRSDQL (SEQ ID NO: 187)1000.000 2 332 KRYFKLSHL (SEQ ID NO: 127) 300.000 3 423 KKFARSDEL (SEQID NO: 122) 150.000 4 390 RKFSRSDHL (SEQ ID NO: 183) 150.000 5 439QRNMTKLQL (SEQ ID NO: 173) 20.000 6 329 GCNKRYFKL (SEQ ID NO: 90) 10.0007 10 ALLPAVPSL (SEQ ID NO: 34) 10.000 8 180 DPMGQQGSL (SEQ ID NO: 59)9.000 9 301 RRVPGVAPT (SEQ ID NO: 189) 6.000 10 126 RMFPNAPYL (SEQ IDNO: 185) 5.000 11 371 KRHQRRHTG (SEQ ID NO: 126) 5.000 12 225 NLYQMTSQL(SEQ ID NO: 147) 5.000 13 144 IRNQGYSTV (SEQ ID NO: 117) 4.000 14 429DELVRHHNM (SEQ ID NO: 53) 3.000 15 437 MHQRNMTKL (SEQ ID NO: 143) 3.00016 125 ARMFPNAPY (SEQ ID NO: 38) 3.000 17 239 NQMNLGATL (SEQ ID NO: 151)3.000 18 286 YRIHTHGVF (SEQ ID NO: 252) 3.000 19 174 HSFKHEDPM (SEQ IDNO: 110) 3.000 20 372 RHQRRHTGV (SEQ ID NO: 181) 3.000

TABLE XII Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B40 (HLA-B40 peptide bindingmotif anchor residues are E at P2, L, W, M, or A at P9; auxiliaryanchors are F, I, or V at P3) Score (Estimate of Half Time of Dis-association of a Molecule Containing Start Subsequence This Sub- RankPosition Residue Listing sequence) 1 81 AEPHEEQCL (SEQ ID NO: 30) 40.0002 429 DELVRHHNM (SEQ ID NO: 53) 24.000 3 410 SEKPFSCRW (SEQ ID NO: 207)20.000 4 318 SEKRPFMCA (SEQ ID NO: 208) 15.000 5 233 LECMTWNQM (SEQ IDNO: 131) 12.000 6 3 SDVRDLNAL (SEQ ID NO: 206) 10.000 7 349 GEKPYQCDF(SEQ ID NO: 91) 8.000 8 6 RDLNALLPA (SEQ ID NO: 177) 5.000 9 85EEQCLSAFT (SEQ ID NO: 65) 4.000 10 315 SETSEKRPF (SEQ ID NO: 209) 4.00011 261 TEGQSNHST (SEQ ID NO: 221) 4.000 12 23 GCALPVSGA (SEQ ID NO: 89)3.000 13 38 LDFAPPGAS (SEQ ID NO: 130) 3.000 14 273 SDNHTTPIL (SEQ IDNO: 204) 2.500 15 206 TDSCTGSQA (SEQ ID NO: 220) 2.500 16 24 CALPVSGAA(SEQ ID NO: 43) 2.000 17 98 GQFTGTAGA (SEQ ID NO: 99) 2.000 18 30GAAQWAPVL (SEQ ID NO: 86) 2.000 19 84 HEEQCLSAF (SEQ ID NO: 107) 2.00020 26 LPVSGAAQW (SEQ ID NO: 138) 2.000

TABLE XIII Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B60 (HLA-B60 peptide bindingmotif anchor residues are E at P2, L at P9; auxiliary anchors are I or Vat P7) Score (Estimate of Half Time of Dis- association of a MoleculeContaining Start Subsequence This Sub- Rank Position Residue Listingsequence) 1 81 AEPHEEQCL (SEQ ID NO: 30) 160.000 2 3 SDVRDLNAL (SEQ IDNO: 206) 40.000 3 429 DELVRHHNM (SEQ ID NO: 53) 40.000 4 233 LECMTWNQM(SEQ ID NO: 131) 22.000 5 273 SDNHTTPIL (SEQ ID NO: 204) 20.000 6 209CTGSQALLL (SEQ ID NO: 52) 8.000 7 30 GAAQWAPVL (SEQ ID NO: 86) 8.000 8318 SEKRPFMCA (SEQ ID NO: 208) 8.000 9 180 DPMGQQGSL (SEQ ID NO: 59)8.000 10 138 LESQPAIRN (SEQ ID NO: 132) 5.280 11 239 NQMNLGATL (SEQ IDNO: 151) 4.400 12 329 GCNKRYFKL (SEQ ID NO: 90) 4.400 13 130 NAPYLPSCL(SEQ ID NO: 144) 4.400 14 85 EEQCLSAFT (SEQ ID NO: 65) 4.400 15 208SCTGSQALL (SEQ ID NO: 202) 4.000 16 207 DSCTGSQAL (SEQ ID NO: 61) 4.00017 218 RTPYSSDNL (SEQ ID NO: 194) 4.000 18 261 TEGQSNHST (SEQ ID NO:221) 4.000 19 18 LGGGGGCAL (SEQ ID NO: 134) 4.000 20 221 YSSDNLYQM (SEQID NO: 253) 2.200

TABLE XIV Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B61 (HLA-B61 peptide bindingmotif anchor residues are E at P2, V at P9; auxiliary anchors are F, I,L, V, Y, W at P3, I at P6) Score (Estimate of Half Time of Dis-association of a Molecule Containing Start Subsequence This Sub- RankPosition Residue Listing sequence) 1 318 SEKRPFMCA (SEQ ID NO: 208)20.000 2 429 DELVRHHNM (SEQ ID NO: 53) 16.000 3 298 QDVRRVPGV (SEQ IDNO: 164) 10.000 4 81 AEPHEEQCL (SEQ ID NO: 30) 8.000 5 233 LECMTWNQM(SEQ ID NO: 131) 8.000 6 6 RDLNALLPA (SEQ ID NO: 177) 5.500 7 85EEQCLSAFT (SEQ ID NO: 65) 4.000 8 261 TEGQSNHST (SEQ ID NO: 221) 4.000 9206 TDSCTGSQA (SEQ ID NO: 220) 2.500 10 295 RGIQDVRRV (SEQ ID NO: 179)2.200 11 3 SDVRDLNAL (SEQ ID NO: 206) 2.000 12 250 VAAGSSSSV (SEQ ID NO:236) 2.000 13 29 SGAAQWAPV (SEQ ID NO: 211) 2.000 14 315 SETSEKRPF (SEQID NO: 209) 1.600 15 138 LESQPAIRN (SEQ ID NO: 132) 1.200 16 244GATLKGVAA (SEQ ID NO: 88) 1.100 17 20 GGGGCALPV (SEQ ID NO: 92) 1.100 18440 RNMTKLQLA (SEQ ID NO: 186) 1.100 19 23 GCALPVSGA (SEQ ID NO: 89)1.100 20 191 QQYSVPPPV (SEQ ID NO: 171) 1.000

TABLE XV Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B62 (HLA-B62 peptide bindingmotif anchor residues are Q or L at P2, F or Y at P9; auxiliary anchorsare I or V at P5) Score (Estimate of Half Time of Dis- association of aMolecule Containing Start Subsequence This Sub- Rank Position ResidueListing sequence) 1 146 NQGYSTVTF (SEQ ID NO: 150) 211.200 2 32AQWAPVLDF (SEQ ID NO: 37) 96.000 3 263 GQSNHSTGY (SEQ ID NO: 100) 96.0004 88 CLSAFTVHF (SEQ ID NO: 48) 96.000 5 17 SLGGGGGCA (SEQ ID NO: 215)9.600 6 239 NQMNLGATL (SEQ ID NO: 151) 8.800 7 191 QQYSVPPPV (SEQ ID NO:171) 8.000 8 98 GQFTGTAGA (SEQ ID NO: 99) 8.000 9 384 QCKTCQRKF (SEQ IDNO: 163) 6.000 10 40 FAPPGASAY (SEQ ID NO: 74) 4.800 11 227 YQMTSQLEC(SEQ ID NO: 251) 4.800 12 187 SLGEQQYSV (SEQ ID NO: 214) 4.400 13 86EQCLSAFTV (SEQ ID NO: 69) 4.400 14 152 VTFDGTPSY (SEQ ID NO: 244) 4.40015 101 TGTAGACRY (SEQ ID NO: 224) 4.000 16 242 NLGATLKGV (SEQ ID NO:146) 4.000 17 92 FTVHFSGQF (SEQ ID NO: 85) 4.000 18 7 DLNALLPAV (SEQ IDNO: 58) 4.000 19 123 GQARMFPNA (SEQ ID NO: 98) 4.000 20 280 ILCGAQYRI(SEQ ID NO: 116) 3.120

TABLE XVI Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B7 (HLA-B7 peptide bindingmotif anchor residues are P at P2, L or F at P9; auxiliary anchor is Rat P2) Score (Estimate of Half Time of Dis- association of a MoleculeContaining Start Subsequence This Sub- Rank Position Residue Listingsequence) 1 180 DPMGQQGSL (SEQ ID NO: 59) 240.000 2 4 DVRDLNALL (SEQ IDNO: 62) 200.000 3 302 RVPGVAPTL (SEQ ID NO: 195) 20.000 4 30 GAAQWAPVL(SEQ ID NO: 86) 12.000 5 239 NQMNLGATL (SEQ ID NO: 151) 12.000 6 130NAPYLPSCL (SEQ ID NO: 144) 12.000 7 10 ALLPAVPSL (SEQ ID NO: 34) 12.0008 299 DVRRVPGVA (SEQ ID NO: 63) 5.000 9 208 SCTGSQALL (SEQ ID NO: 202)4.000 10 303 VPGVAPTLV (SEQ ID NO: 242) 4.000 11 18 LGGGGGCAL (SEQ IDNO: 134) 4.000 12 218 RTPYSSDNL (SEQ ID NO: 194) 4.000 13 207 DSCTGSQAL(SEQ ID NO: 61) 4.000 14 209 CTGSQALLL (SEQ ID NO: 52) 4.000 15 329GCNKRYFKL (SEQ ID NO: 90) 4.000 16 235 CMTWNQMNL (SEQ ID NO: 49) 4.00017 441 NMTKLQLAL (SEQ ID NO: 149) 4.000 18 126 RMFPNAPYL (SEQ ID NO:185) 4.000 19 225 NLYQMTSQL (SEQ ID NO: 147) 4.000 20 143 AIRNQGYST (SEQID NO: 33) 3.000

TABLE XVII Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B8 (HLA-B8 peptide bindingmotif anchor residues are K at P3, K or R at P5, L at P9) Score(Estimate of Half Time of Dis- association of a Molecule ContainingStart Subsequence This Sub- Rank Position Residue Listing sequence) 1329 GCNKRYFKL (SEQ ID NO: 90) 16.000 2 4 DVRDLNALL (SEQ ID NO: 62)12.000 3 316 ETSEKRPFM (SEQ ID NO: 73) 3.000 4 180 DPMGQQGSL (SEQ ID NO:59) 1.600 5 208 SCTGSQALL (SEQ ID NO: 202) 0.800 6 130 NAPYLPSCL (SEQ IDNO: 144) 0.800 7 244 GATLKGVAA (SEQ ID NO: 88) 0.800 8 30 GAAQWAPVL (SEQID NO: 86) 0.800 9 299 DVRRVPGVA (SEQ ID NO: 63) 0.400 10 420 SCQKKFARS(SEQ ID NO: 200) 0.400 11 387 TCQRKFSRS (SEQ ID NO: 219) 0.400 12 225NLYQMTSQL (SEQ ID NO: 147) 0.400 13 141 QPAIRNQGY (SEQ ID NO: 170) 0.40014 10 ALLPAVPSL (SEQ ID NO: 34) 0.400 15 207 DSCTGSQAL (SEQ ID NO: 61)0.400 16 384 QCKTCQRKF (SEQ ID NO: 163) 0.400 17 136 SCLESQPAI (SEQ IDNO: 198) 0.300 18 347 HTGEKPYQC (SEQ ID NO: 112) 0.300 19 401 HTRTHTGKT(SEQ ID NO: 114) 0.200 20 332 KRYFKLSHL (SEQ ID NO: 127) 0.200

TABLE XVIII Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B 2702 (HLA-B2702 peptidebinding motif anchor residues are R at P2; F, Y, I, L, or W at P9) Score(Estimate of Half Time of Dis- association of a Molecule ContainingStart Subsequence This Sub- Rank Position Residue Listing sequence) 1332 KRYFKLSHL (SEQ ID NO: 127) 900.000 2 362 RRFSRSDQL (SEQ ID NO: 187)900.000 3 286 YRIHTHGVF (SEQ ID NO: 252) 200.000 4 125 ARMFPNAPY (SEQ IDNO: 38) 200.000 5 375 RRHTGVKPF (SEQ ID NO: 188) 180.000 6 32 AQWAPVLDF(SEQ ID NO: 37) 100.000 7 301 RRVPGVAPT (SEQ ID NO: 189) 60.000 8 439QRNMTKLQL (SEQ ID NO: 173) 60.000 9 126 RMFPNAPYL (SEQ ID NO: 185)22.500 10 426 ARSDELVRH (SEQ ID NO: 39) 20.000 11 146 NQGYSTVTF (SEQ IDNO: 150) 20.000 12 144 IRNQGYSTV (SEQ ID NO: 117) 20.000 13 389QRKFSRSDH (SEQ ID NO: 172) 20.000 14 263 GQSNHSTGY (SEQ ID NO: 100)20.000 15 416 CRWPSCQKK (SEQ ID NO: 50) 20.000 16 191 QQYSVPPPV (SEQ IDNO: 171) 10.000 17 217 LRTPYSSDN (SEQ ID NO: 140) 10.000 18 107CRYGPFGPP (SEQ ID NO: 51) 10.000 19 98 GQFTGTAGA (SEQ ID NO: 99) 10.00020 239 NQMNLGATL (SEQ ID NO: 151) 6.000

TABLE XIX Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B 2705 (HLA-B2705 peptidebinding motif anchor residues are R at P2; L or F at P9; Y, R, H, and K,have also been found at P9 for naturally processed epitopes) Score(Estimate of Half Time of Dis- association of a Molecule ContainingStart Subsequence This Sub- Rank Position Residue Listing sequence) 1332 KRYFKLSHL (SEQ ID NO: 127) 30000.000 2 362 RRFSRSDQL (SEQ ID NO:187) 30000.000 3 416 CRWPSCQKK (SEQ ID NO: 50) 10000.000 4 439 QRNMTKLQL(SEQ ID NO: 173) 2000.000 5 286 YRIHTHGVF (SEQ ID NO: 252) 1000.000 6125 ARMFPNAPY (SEQ ID NO: 38) 1000.000 7 294 FRGIQDVRR (SEQ ID NO: 81)1000.000 8 432 VRHHNMHQR (SEQ ID NO: 243) 1000.000 9 169 AQFPNHSFK (SEQID NO: 36) 1000.000 10 375 RRHTGVKPF (SEQ ID NO: 188) 900.000 11 126RMFPNAPYL (SEQ ID NO: 185) 750.000 12 144 IRNQGYSTV (SEQ ID NO: 117)600.000 13 301 RRVPGVAPT (SEQ ID NO: 189) 600.000 14 32 AQWAPVLDF (SEQID NO: 37) 500.000 15 191 QQYSVPPPV (SEQ ID NO: 171) 300.000 16 373HQRRHTGVK (SEQ ID NO: 109) 200.000 17 426 ARSDELVRH (SEQ ID NO: 39)200.000 18 383 FQCKTCQRK (SEQ ID NO: 80) 200.000 19 239 NQMNLGATL (SEQID NO: 151) 200.000 20 389 QRKFSRSDH (SEQ ID NO: 172) 200.000

TABLE XX Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B 3501 (HLA-B3501 peptidebinding motif anchor residues are P at P2; Y, F, M, L, or I at P9) Score(Estimate of Half Time of Dis- association of a Molecule ContainingStart Subsequence This Sub- Rank Position Residue Listing sequence) 1278 TPILCGAQY (SEQ ID NO: 227) 40.000 2 141 QPAIRNQGY (SEQ ID NO: 170)40.000 3 219 TPYSSDNLY (SEQ ID NO: 231) 40.000 4 327 YPGCNKRYF (SEQ IDNO: 250) 20.000 5 163 TPSHHAAQF (SEQ ID NO: 228) 20.000 6 180 DPMGQQGSL(SEQ ID NO: 59) 20.000 7 221 YSSDNLYQM (SEQ ID NO: 253) 20.000 8 26LPVSGAAQW (SEQ ID NO: 138) 10.000 9 174 HSFKHEDPM (SEQ ID NO: 110)10.000 10 82 EPHEEQCLS (SEQ ID NO: 68) 6.000 11 213 QALLLRTPY (SEQ IDNO: 160) 6.000 12 119 QASSGQARM (SEQ ID NO: 161) 6.000 13 4 DVRDLNALL(SEQ ID NO: 62) 6.000 14 40 FAPPGASAY (SEQ ID NO: 74) 6.000 15 120ASSGQARMF (SEQ ID NO: 40) 5.000 16 207 DSCTGSQAL (SEQ ID NO: 61) 5.00017 303 VPGVAPTLV (SEQ ID NO: 242) 4.000 18 316 ETSEKRPFM (SEQ ID NO: 73)4.000 19 152 VTFDGTPSY (SEQ ID NO: 244) 4.000 20 412 KPFSCRWPS (SEQ IDNO: 123) 4.000

TABLE XXI Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B 3701 (HLA-B3701 peptidebinding motif anchor residues are F, M, or L at P8; I or L at P9;auxiliary anchors are D, E at P2 and V, I at P5) Score (Estimate of HalfTime of Dis- association of a Molecule Containing Start Subsequence ThisSub- Rank Position Residue Listing sequence) 1 3 SDVRDLNAL (SEQ ID NO:206) 40.000 2 273 SDNHTTPIL (SEQ ID NO: 204) 40.000 3 81 AEPHEEQCL (SEQID NO: 30) 10.000 4 298 QDVRRVPGV (SEQ ID NO: 164) 8.000 5 428 SDELVRHHN(SEQ ID NO: 203) 6.000 6 85 EEQCLSAFT (SEQ ID NO: 65) 5.000 7 208SCTGSQALL (SEQ ID NO: 202) 5.000 8 4 DVRDLNALL (SEQ ID NO: 62) 5.000 9209 CTGSQALLL (SEQ ID NO: 52) 5.000 10 38 LDFAPPGAS (SEQ ID NO: 130)4.000 11 223 SDNLYQMTS (SEQ ID NO: 205) 4.000 12 179 EDPMGQQGS (SEQ IDNO: 64) 4.000 13 206 TDSCTGSQA (SEQ ID NO: 220) 4.000 14 6 RDLNALLPA(SEQ ID NO: 177) 4.000 15 84 HEEQCLSAF (SEQ ID NO: 107) 2.000 16 233LECMTWNQM (SEQ ID NO: 131) 2.000 17 429 DELVRHHNM (SEQ ID NO: 53) 2.00018 315 SETSEKRPF (SEQ ID NO: 209) 2.000 19 349 GEKPYQCDF (SEQ ID NO: 91)2.000 20 302 RVPGVAPTL (SEQ ID NO: 195) 1.500

TABLE XXII Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B 3801 (HLA-B3801 peptidebinding motif anchor residues are F or L at P9; Auxiliary anchors are Hat P2 and D or E at P3) Score (Estimate of Half Time of Dis- associationof a Molecule Containing Start Subsequence This Sub- Rank PositionResidue Listing sequence) 1 437 MHQRNMTKL (SEQ ID NO: 143) 36.000 2 434HHNMHQRNM (SEQ ID NO: 108) 6.000 3 372 RHQRRHTGV (SEQ ID NO: 181) 6.0004 180 DPMGQQGSL (SEQ ID NO: 59) 4.000 5 433 RHHNMHQRN (SEQ ID NO: 180)3.900 6 165 SHHAAQFPN (SEQ ID NO: 213) 3.900 7 202 CHTPTDSCT (SEQ ID NO:45) 3.000 8 396 DHLKTHTRT (SEQ ID NO: 57) 3.000 9 161 GHTPSHHAA (SEQ IDNO: 94) 3.000 10 302 RVPGVAPTL (SEQ ID NO: 195) 2.600 11 417 RWPSCQKKF(SEQ ID NO: 196) 2.400 12 327 YPGCNKRYF (SEQ ID NO: 250) 2.400 13 208SCTGSQALL (SEQ ID NO: 202) 2.000 14 163 TPSHHAAQF (SEQ ID NO: 228) 2.00015 120 ASSGQARMF (SEQ ID NO: 40) 2.000 16 18 LGGGGGCAL (SEQ ID NO: 134)2.000 17 177 KHEDPMGQQ (SEQ ID NO: 121) 1.800 18 83 PHEEQCLSA (SEQ IDNO: 154) 1.800 19 10 ALLPAVPSL (SEQ ID NO: 34) 1.300 20 225 NLYQMTSQL(SEQ ID NO: 147) 1.300

TABLE XXIII Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B 3901 (HLA-B3901 peptidebinding motif anchor residues are R or H at P2; L at P9; auxiliaryanchors are I, V, or L at P6) Score (Estimate of Half Time of Dis-association of a Molecule Containing Start Subsequence This Sub- RankPosition Residue Listing sequence) 1 437 MHQRNMTKL (SEQ ID NO: 143)135.000 2 332 KRYFKLSHL (SEQ ID NO: 127) 45.000 3 434 HHNMHQRNM (SEQ IDNO: 108) 30.000 4 362 RRFSRSDQL (SEQ ID NO: 187) 30.000 5 372 RHQRRHTGV(SEQ ID NO: 181) 30.000 6 10 ALLPAVPSL (SEQ ID NO: 34) 9.000 7 439QRNMTKLQL (SEQ ID NO: 173) 7.500 8 390 RKFSRSDHL (SEQ ID NO: 183) 6.0009 396 DHLKTHTRT (SEQ ID NO: 57) 6.000 10 239 NQMNLGATL (SEQ ID NO: 151)6.000 11 423 KKFARSDEL (SEQ ID NO: 122) 6.000 12 126 RMFPNAPYL (SEQ IDNO: 185) 6.000 13 225 NLYQMTSQL (SEQ ID NO: 147) 6.000 14 180 DPMGQQGSL(SEQ ID NO: 59) 6.000 15 144 IRNQGYSTV (SEQ ID NO: 117) 5.000 16 136SCLESQPAI (SEQ ID NO: 198) 4.000 17 292 GVFRGIQDV (SEQ ID NO: 103) 3.00018 302 RVPGVAPTL (SEQ ID NO: 195) 3.000 19 208 SCTGSQALL (SEQ ID NO:202) 3.000 20 207 DSCTGSQAL (SEQ ID NO: 61) 3.000

TABLE XXIV Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B 3902 (HLA-B3902 peptidebinding motif anchor residues are K or Q at P2; L at P9; auxiliaryanchors are I, L, F, or V at P5) Score (Estimate of Half Time ofDisassociation of a Molecule Containing Start Subsequence This RankPosition Residue Listing Subsequence) 1 239 NQMNLGATL (SEQ ID NO: 24.000151) 2 390 RKFSRSDHL (SEQ ID NO: 20.000 183) 3 423 KKFARSDEL (SEQ ID NO:20.000 122) 4 32 AQWAPVLDF (SEQ ID NO: 5.000 37) 5 146 NQGYSTVTF (SEQ IDNO: 5.000 150) 6 130 NAPYLPSCL (SEQ ID NO: 2.400 144) 7 225 NLYQMTSQL(SEQ ID NO: 2.400 147) 8 30 GAAQWAPVL (SEQ ID NO: 2.400 86) 9 441NMTKLQLAL (SEQ ID NO: 2.400 149) 10 302 RVPGVAPTL (SEQ ID NO: 2.400 195)11 126 RMFPNAPYL (SEQ ID NO: 2.000 185) 12 218 RTPYSSDNL (SEQ ID NO:2.000 194) 13 209 CTGSQALLL (SEQ ID NO: 2.000 52) 14 332 KRYFKLSHL (SEQID NO: 2.000 127) 15 180 DPMGQQGSL (SEQ ID NO: 2.000 59) 16 437MHQRNMTKL (SEQ ID NO: 2.000 143) 17 207 DSCTGSQAL (SEQ ID NO: 2.000 61)18 208 SCTGSQALL (SEQ ID NO: 2.000 202) 19 329 GCNKRYFKL (SEQ ID NO:2.000 90) 20 10 ALLPAVPSL (SEQ ID NO: 2.000 34)

TABLE XXV Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B 4403 (HLA-B4403 peptidebinding motif anchor residues are E at P2; Y or F at P9) Score (Estimateof Half Time of Disassociation of Start Subsequence a MoleculeContaining Rank Position Residue Listing This Subsequence) 1 315SETSEKRPF 80.000 (SEQ ID NO: 209) 2 349 GEKPYQCDF 80.000 (SEQ ID NO: 91)3 84 HEEQCLSAF 60.000 (SEQ ID NO: 107) 4 410 SEKPFSCRW 48.000 (SEQ IDNO: 207) 5 429 DELVRHHNM 24.000 (SEQ ID NO: 53) 6 278 TPILCGAQY 15.000(SEQ ID NO: 227) 7 141 QPAIRNQGY 9.000 (SEQ ID NO: 170) 8 40 FAPPGASAY9.000 (SEQ ID NO: 74) 9 213 QALLLRTPY 9.000 (SEQ ID NO: 160) 10 318SEKRPFMCA 8.000 (SEQ ID NO: 208) 11 81 AEPHEEQCL 8.000 (SEQ ID NO: 30)12 152 VTFDGTPSY 4.500 (SEQ ID NO: 244) 13 101 TGTAGACRY 4.500 (SEQ IDNO: 224) 14 120 ASSGQARMF 4.500 (SEQ ID NO: 40) 15 261 TEGQSNHST 4.000(SEQ ID NO: 221) 16 85 EEQCLSAFT 4.000 (SEQ ID NO: 65) 17 233 LECMTWNQM4.000 (SEQ ID NO: 131) 18 104 AGACRYGPF 4.000 (SEQ ID NO: 31) 19 3SDVRDLNAL 3.000 (SEQ ID NO: 206) 20 185 QGSLGEQQY 3.000 (SEQ ID NO: 166)

TABLE XXVI Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B 5101 (HLA-B5101 peptidebinding motif anchor residues are A, P, or G at P2; F or I at P9) Score(Estimate of Half Time of Disassociation of Start Subsequence a MoleculeContaining Rank Position Residue Listing This Subsequence) 1 303VPGVAPTLV 314.600 (SEQ ID NO: 242) 2 180 DPMGQQGSL 242.000 (SEQ ID NO:59) 3 250 VAAGSSSSV 157.300 (SEQ ID NO: 236) 4 130 NAPYLPSCL 50.000 (SEQID NO: 144) 5 30 GAAQWAPVL 50.000 (SEQ ID NO: 86) 6 20 GGGGCALPV 44.000(SEQ ID NO: 92) 7 64 PPPPPHSFI 40.000 (SEQ ID NO: 157) 8 29 SGAAQWAPV40.000 (SEQ ID NO: 211) 9 18 LGGGGGCAL 31.460 (SEQ ID NO: 134) 10 295RGIQDVRRV 22.000 (SEQ ID NO: 179) 11 119 QASSGQARM 18.150 (SEQ ID NO:161) 12 418 WPSCQKKFA 12.100 (SEQ ID NO: 246) 13 82 EPHEEQCLS 12.100(SEQ ID NO: 68) 14 110 GPFGPPPPS 11.000 (SEQ ID NO: 96) 15 272 ESDNHTTPI8.000 (SEQ ID NO: 71) 16 306 VAPTLVRSA 7.150 (SEQ ID NO: 237) 17 280ILCGAQYRI 6.921 (SEQ ID NO: 116) 18 219 TPYSSDNLY 6.600 (SEQ ID NO: 231)19 128 FPNAPYLPS 6.500 (SEQ ID NO: 79) 20 204 TPTDSCTGS 6.050 (SEQ IDNO: 230)

TABLE XXVII Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B 5102 (HLA-B5102 peptidebinding motif anchor residues are P, A, or G at P2; I or V at P9;auxiliary anchor is Y at P3) Score (Estimate of Half Time ofDisassociation of Start Subsequence a Molecule Containing Rank PositionResidue Listing This Subsequence) 1 295 RGIQDVRRV 290.400 (SEQ ID NO:179) 2 303 VPGVAPTLV 200.000 (SEQ ID NO: 242) 3 180 DPMGQQGSL 133.100(SEQ ID NO: 59) 4 250 VAAGSSSSV 110.000 (SEQ ID NO: 236) 5 30 GAAQWAPVL55.000 (SEQ ID NO: 86) 6 130 NAPYLPSCL 50.000 (SEQ ID NO: 144) 7 20GGGGCALPV 44.000 (SEQ ID NO: 92) 8 29 SGAAQWAPV 44.000 (SEQ ID NO: 211)9 64 PPPPPHSFI 40.000 (SEQ ID NO: 157) 10 119 QASSGQARM 36.300 (SEQ IDNO: 161) 11 110 GPFGPPPPS 27.500 (SEQ ID NO: 96) 12 412 KPFSCRWPS 25.000(SEQ ID NO: 123) 13 18 LGGGGGCAL 24.200 (SEQ ID NO: 134) 14 24 CALPVSGAA16.500 (SEQ ID NO: 43) 15 219 TPYSSDNLY 15.000 (SEQ ID NO: 231) 16 292GVFRGIQDV 14.641 (SEQ ID NO: 103) 17 136 SCLESQPAI 14.520 (SEQ ID NO:198) 18 418 WPSCQKKFA 12.100 (SEQ ID NO: 246) 19 269 TGYESDNHT 11.000(SEQ ID NO: 225) 20 351 KPYQCDFKD 11.000 (SEQ ID NO: 124)

TABLE XXVIII Results of BIMAS HLA Peptide Binding Prediction Analysisfor Binding of Human WT1 Peptides to Human HLA B 5201 (HLA-B5201 peptidebinding motif anchor residues are I or V at P8; I or V at P9; auxiliaryanchors are Q at P2, F, Y, or W at P3, L, I, or V at P5) Score (Estimateof Half Time of Disassociation of Start Subsequence a MoleculeContaining Rank Position Residue Listing This Subsequence) 1 191QQYSVPPPV 100.000 (SEQ ID NO: 171) 2 32 AQWAPVLDF 30.000 (SEQ ID NO: 37)3 243 LGATLKGVA 16.500 (SEQ ID NO: 133) 4 303 VPGVAPTLV 13.500 (SEQ IDNO: 242) 5 86 EQCLSAFTV 12.000 (SEQ ID NO: 69) 6 295 RGIQDVRRV 10.000(SEQ ID NO: 179) 7 98 GQFTGTAGA 8.250 (SEQ ID NO: 99) 8 292 GVFRGIQDV8.250 (SEQ ID NO: 103) 9 29 SGAAQWAPV 6.000 (SEQ ID NO: 211) 10 146NQGYSTVTF 5.500 (SEQ ID NO: 150) 11 20 GGGGCALPV 5.000 (SEQ ID NO: 92)12 239 NQMNLGATL 4.000 (SEQ ID NO: 151) 13 64 PPPPPHSFI 3.600 (SEQ IDNO: 157) 14 273 SDNHTTPIL 3.300 (SEQ ID NO: 204) 15 286 YRIHTHGVF 3.000(SEQ ID NO: 252) 16 269 TGYESDNHT 3.000 (SEQ ID NO: 225) 17 406TGKTSEKPF 2.750 (SEQ ID NO: 222) 18 327 YPGCNKRYF 2.750 (SEQ ID NO: 250)19 7 DLNALLPAV 2.640 (SEQ ID NO: 58) 20 104 AGACRYGPF 2.500 (SEQ ID NO:31)

TABLE XXIX Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA B 5801 (HLA-B5801 peptidebinding motif anchor residues are A, S, or T at P2; F or W at P9;auxiliary anchors are P, E, or K at P4, V, I, L, M, of F, at P5) Score(Estimate of Half Time of Disassociation of Start Subsequence a MoleculeContaining Rank Position Residue Listing This Subsequence) 1 230TSQLECMTW 96.800 (SEQ ID NO: 234) 2 92 FTVHFSGQF 60.000 (SEQ ID NO: 85)3 120 ASSGQARMF 40.000 (SEQ ID NO: 40) 4 168 AAQFPNHSF 20.000 (SEQ IDNO: 29) 5 408 KTSEKPFSC 12.000 (SEQ ID NO: 129) 6 394 RSDHLKTHT 9.900(SEQ ID NO: 192) 7 276 HTTPILCGA 7.200 (SEQ ID NO: 115) 8 218 RTPYSSDNL6.600 (SEQ ID NO: 194) 9 152 VTFDGTPSY 6.000 (SEQ ID NO: 244) 10 40FAPPGASAY 6.000 (SEQ ID NO: 74) 11 213 QALLLRTPY 4.500 (SEQ ID NO: 160)12 347 HTGEKPYQC 4.400 (SEQ ID NO: 112) 13 252 AGSSSSVKW 4.400 (SEQ IDNO: 32) 14 211 GSQALLLRT 4.356 (SEQ ID NO: 102) 15 174 HSFKHEDPM 4.000(SEQ ID NO: 110) 16 317 TSEKRPFMC 4.000 (SEQ ID NO: 233) 17 26 LPVSGAAQW4.000 (SEQ ID NO: 138) 18 289 HTHGVFRGI 3.600 (SEQ ID NO: 113) 19 222SSDNLYQMT 3.300 (SEQ ID NO: 217) 20 96 FSGQFTGTA 3.300 (SEQ ID NO: 82)

TABLE XXX Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA CW0301 (HLA-CW0301 peptidebinding motif anchor residues are L, F, M, or I at P9; auxiliary anchorsare V, I, Y, L, or M, at P3, P at P4, F or Y at P6) Score (Estimate ofHalf Time of Disassociation of Start Subsequence a Molecule ContainingRank Position Residue Listing This Subsequence) 1 10 ALLPAVPSL 100.000(SEQ ID NO: 34) 2 332 KRYFKLSHL 48.000 (SEQ ID NO: 127) 3 126 RMFPNAPYL36.000 (SEQ ID NO: 185) 4 3 SDVRDLNAL 30.000 (SEQ ID NO: 206) 5 239NQMNLGATL 24.000 (SEQ ID NO: 151) 6 225 NLYQMTSQL 24.000 (SEQ ID NO:147) 7 180 DPMGQQGSL 20.000 (SEQ ID NO: 59) 8 362 RRFSRSDQL 12.000 (SEQID NO: 187) 9 329 GCNKRYFKL 10.000 (SEQ ID NO: 90) 10 286 YRIHTHGVF10.000 (SEQ ID NO: 252) 11 301 RRVPGVAPT 10.000 (SEQ ID NO: 189) 12 24CALPVSGAA 10.000 (SEQ ID NO: 43) 13 136 SCLESQPAI 7.500 (SEQ ID NO: 198)14 437 MHQRNMTKL 7.200 (SEQ ID NO: 143) 15 390 RKFSRSDHL 6.000 (SEQ IDNO: 183) 16 423 KKFARSDEL 6.000 (SEQ ID NO: 122) 17 92 FTVHFSGQF 5.000(SEQ ID NO: 85) 18 429 DELVRHHNM 5.000 (SEQ ID NO: 53) 19 130 NAPYLPSCL4.800 (SEQ ID NO: 144) 20 30 GAAQWAPVL 4.000 (SEQ ID NO: 86)

TABLE XXXI Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA CW0401 (HLA-CW0401 peptidebinding motif anchor residues are Y, P, or F at P2; L, F, or M at P9;auxiliary anchors are V, I, or L at P6) Score (Estimate of Half Time ofDisassociation of Start Subsequence a Molecule Containing Rank PositionResidue Listing This Subsequence) 1 356 DFKDCERRF 120.000 (SEQ ID NO:55) 2 334 YFKLSHLQM 100.000 (SEQ ID NO: 248) 3 180 DPMGQQGSL 88.000 (SEQID NO: 59) 4 163 TPSHHAAQF 52.800 (SEQ ID NO: 228) 5 327 YPGCNKRYF40.000 (SEQ ID NO: 250) 6 285 QYRIHTHGV 27.500 (SEQ ID NO: 175) 7 424KFARSDELV 25.000 (SEQ ID NO: 119) 8 326 AYPGCNKRY 25.000 (SEQ ID NO: 42)9 192 QYSVPPPVY 25.000 (SEQ ID NO: 176) 10 417 RWPSCQKKF 22.000 (SEQ IDNO: 196) 11 278 TPILCGAQY 12.000 (SEQ ID NO: 227) 12 10 ALLPAVPSL 11.616(SEQ ID NO: 34) 13 141 QPAIRNQGY 11.000 (SEQ ID NO: 170) 14 303VPGVAPTLV 11.000 (SEQ ID NO: 242) 15 219 TPYSSDNLY 10.000 (SEQ ID NO:231) 16 39 DFAPPGASA 7.920 (SEQ ID NO: 54) 17 99 QFTGTAGAC 6.000 (SEQ IDNO: 165) 18 4 DVRDLNALL 5.760 (SEQ ID NO: 62) 19 70 SFIKQEPSW 5.500 (SEQID NO: 210) 20 63 PPPPPPHSF 5.280 (SEQ ID NO: 158)

TABLE XXXII Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Human HLA CW0602 (HLA-CW0602 peptidebinding motif anchor residues are L, I, V, or Y at P9; auxiliary anchorsare I, L, F, or M at P5, V, I, or L at P6) Score (Estimate of Half Timeof Disassociation of Start Subsequence a Molecule Containing RankPosition Residue Listing This Subsequence) 1 332 KRYFKLSHL 9.680 (SEQ IDNO: 127) 2 239 NQMNLGATL 6.600 (SEQ ID NO: 151) 3 130 NAPYLPSCL 6.600(SEQ ID NO: 144) 4 7 DLNALLPAV 6.000 (SEQ ID NO: 58) 5 441 NMTKLQLAL6.000 (SEQ ID NO: 149) 6 225 NLYQMTSQL 6.000 (SEQ ID NO: 147) 7 4DVRDLNALL 6.000 (SEQ ID NO: 62) 8 3 SDVRDLNAL 4.400 (SEQ ID NO: 206) 910 ALLPAVPSL 4.000 (SEQ ID NO: 34) 10 213 QALLLRTPY 3.300 (SEQ ID NO:160) 11 319 EKRPFMCAY 3.000 (SEQ ID NO: 67) 12 30 GAAQWAPVL 2.200 (SEQID NO: 86) 13 242 NLGATLKGV 2.200 (SEQ ID NO: 146) 14 292 GVFRGIQDV2.200 (SEQ ID NO: 103) 15 207 DSCTGSQAL 2.200 (SEQ ID NO: 61) 16 362RRFSRSDQL 2.200 (SEQ ID NO: 187) 17 439 QRNMTKLQL 2.200 (SEQ ID NO: 173)18 295 RGIQDVRRV 2.200 (SEQ ID NO: 179) 19 423 KKFARSDEL 2.200 (SEQ IDNO: 122) 20 180 DPMGQQGSL 2.200 (SEQ ID NO: 59)

TABLE XXXIII Results of BIMAS HLA Peptide Binding Prediction Analysisfor Binding of Human WT1 Peptides to Human HLA CW0702 (HLA-CW0702peptide binding motif anchor residues are Y, F, or L at P9; auxiliaryanchors are Y or P at P2, V, Y, I, L, F, or M at P5, V, I, L, or M atP6) Score (Estimate of Half Time of Disassociation of Start Subsequencea Molecule Containing Rank Position Residue Listing This Subsequence) 1319 EKRPFMCAY 26.880 (SEQ ID NO: 67) 2 326 AYPGCNKRY 24.000 (SEQ ID NO:42) 3 40 FAPPGASAY 14.784 (SEQ ID NO: 74) 4 192 QYSVPPPVY 12.000 (SEQ IDNO: 176) 5 278 TPILCGAQY 12.000 (SEQ ID NO: 227) 6 219 TPYSSDNLY 12.000(SEQ ID NO: 231) 7 213 QALLLRTPY 8.800 (SEQ ID NO: 160) 8 125 ARMFPNAPY8.000 (SEQ ID NO: 38) 9 327 YPGCNKRYF 6.600 (SEQ ID NO: 250) 10 152VTFDGTPSY 5.600 (SEQ ID NO: 244) 11 141 QPAIRNQGY 4.800 (SEQ ID NO: 170)12 345 RKHTGEKPY 4.000 (SEQ ID NO: 184) 13 185 QGSLGEQQY 4.000 (SEQ IDNO: 166) 14 101 TGTAGACRY 4.000 (SEQ ID NO: 224) 15 375 RRHTGVKPF 4.000(SEQ ID NO: 188) 16 263 GQSNHSTGY 4.000 (SEQ ID NO: 100) 17 163TPSHHAAQF 3.000 (SEQ ID NO: 228) 18 33 QWAPVLDFA 2.688 (SEQ ID NO: 174)19 130 NAPYLPSCL 2.640 (SEQ ID NO: 144) 20 84 HEEQCLSAF 2.400 (SEQ IDNO: 107)

TABLE XXXIV Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Mouse MHC Class I Db Score (Estimate ofHalf Time of Disassociation of Start Subsequence a Molecule ContainingRank Position Residue Listing This Subsequence) 1 235 CMTWNQMNL 5255.712(SEQ ID NO: 49) 2 126 RMFPNAPYL 1990.800 (SEQ ID NO: 185) 3 221YSSDNLYQM 930.000 (SEQ ID NO: 253) 4 228 QMTSQLECM 33.701 (SEQ ID NO:169) 5 239 NQMNLGATL 21.470 (SEQ ID NO: 151) 6 441 NMTKLQLAL 19.908 (SEQID NO: 149) 7 437 MHQRNMTKL 19.837 (SEQ ID NO: 143) 8 136 SCLESQPAI11.177 (SEQ ID NO: 198) 9 174 HSFKHEDPM 10.800 (SEQ ID NO: 110) 10 302RVPGVAPTL 10.088 (SEQ ID NO: 195) 11 130 NAPYLPSCL 8.400 (SEQ ID NO:144) 12 10 ALLPAVPSL 5.988 (SEQ ID NO: 34) 13 208 SCTGSQALL 4.435 (SEQID NO: 202) 14 209 CTGSQALLL 3.548 (SEQ ID NO: 52) 15 238 WNQMNLGAT3.300 (SEQ ID NO: 245) 16 218 RTPYSSDNL 3.185 (SEQ ID NO: 194) 17 24CALPVSGAA 2.851 (SEQ ID NO: 43) 18 18 LGGGGGCAL 2.177 (SEQ ID NO: 134)19 142 PAIRNQGYS 2.160 (SEQ ID NO: 152) 20 30 GAAQWAPVL 1.680 (SEQ IDNO: 86)

TABLE XXXV Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Mouse MHC Class I Dd Score (Estimate ofHalf Time of Disassociation of Start Subsequence a Molecule ContainingRank Position Residue Listing This Subsequence) 1 112 FGPPPPSQA 48.000(SEQ ID NO: 76) 2 122 SGQARMFPN 36.000 (SEQ ID NO: 212) 3 104 AGACRYGPF30.000 (SEQ ID NO: 31) 4 218 RTPYSSDNL 28.800 (SEQ ID NO: 194) 5 130NAPYLPSCL 20.000 (SEQ ID NO: 144) 6 302 RVPGVAPTL 20.000 (SEQ ID NO:195) 7 18 LGGGGGCAL 20.000 (SEQ ID NO: 134) 8 81 AEPHEEQCL 10.000 (SEQID NO: 30) 9 29 SGAAQWAPV 7.200 (SEQ ID NO: 211) 10 423 KKFARSDEL 7.200(SEQ ID NO: 122) 11 295 RGIQDVRRV 7.200 (SEQ ID NO: 179) 12 390RKFSRSDHL 6.000 (SEQ ID NO: 183) 13 332 KRYFKLSHL 6.000 (SEQ ID NO: 127)14 362 RRFSRSDQL 6.000 (SEQ ID NO: 187) 15 417 RWPSCQKKF 6.000 (SEQ IDNO: 196) 16 160 YGHTPSHHA 6.000 (SEQ ID NO: 249) 17 20 GGGGCALPV 6.000(SEQ ID NO: 92) 18 329 GCNKRYFKL 5.000 (SEQ ID NO: 90) 19 372 RHQRRHTGV4.500 (SEQ ID NO: 181) 20 52 GGPAPPPAP 4.000 (SEQ ID NO: 93)

TABLE XXXVI Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Mouse MHC Class I Kb Score (Estimate ofHalf Time of Disassociation of Start Subsequence a Molecule ContainingRank Position Residue Listing This Subsequence) 1 329 GCNKRYFKL 24.000(SEQ ID NO: 90) 2 225 NLYQMTSQL 10.000 (SEQ ID NO: 147) 3 420 SCQKKFARS3.960 (SEQ ID NO: 200) 4 218 RTPYSSDNL 3.630 (SEQ ID NO: 194) 5 437MHQRNMTKL 3.600 (SEQ ID NO: 143) 6 387 TCQRKFSRS 3.600 (SEQ ID NO: 219)7 302 RVPGVAPTL 3.300 (SEQ ID NO: 195) 8 130 NAPYLPSCL 3.000 (SEQ ID NO:144) 9 289 HTHGVFRGI 3.000 (SEQ ID NO: 113) 10 43 PGASAYGSL 2.400 (SEQID NO: 153) 11 155 DGTPSYGHT 2.400 (SEQ ID NO: 56) 12 273 SDNHTTPIL2.200 (SEQ ID NO: 204) 13 126 RMFPNAPYL 2.200 (SEQ ID NO: 185) 14 128FPNAPYLPS 2.000 (SEQ ID NO: 79) 15 3 SDVRDLNAL 1.584 (SEQ ID NO: 206) 16207 DSCTGSQAL 1.584 (SEQ ID NO: 61) 17 332 KRYFKLSHL 1.500 (SEQ ID NO:127) 18 18 LGGGGGCAL 1.320 (SEQ ID NO: 134) 19 233 LECMTWNQM 1.320 (SEQID NO: 131) 20 441 NMTKLQLAL 1.200 (SEQ ID NO: 149)

TABLE XXXVII Results of BIMAS HLA Peptide Binding Prediction Analysisfor Binding of Human WT1 Peptides to Mouse MHC Class I Kd Score(Estimate of Half Time of Disassociation of Start Subsequence a MoleculeContaining Rank Position Residue Listing This Subsequence) 1 285QYRIHTHGV 600.000 (SEQ ID NO: 175) 2 424 KFARSDELV 288.000 (SEQ ID NO:119) 3 334 YFKLSHLQM 120.000 (SEQ ID NO: 248) 4 136 SCLESQPTI 115.200(SEQ ID NO: 199) 5 239 NQMNLGATL 115.200 (SEQ ID NO: 151) 6 10 ALLPAVSSL115.200 (SEQ ID NO: 35) 7 47 AYGSLGGPA 86.400 (SEQ ID NO: 41) 8 180DPMGQQGSL 80.000 (SEQ ID NO: 59) 9 270 GYESDNHTA 72.000 (SEQ ID NO: 105)10 326 AYPGCNKRY 60.000 (SEQ ID NO: 42) 11 192 QYSVPPPVY 60.000 (SEQ IDNO: 176) 12 272 ESDNHTAPI 57.600 (SEQ ID NO: 70) 13 289 HTHGVFRGI 57.600(SEQ ID NO: 113) 14 126 DVRDLNALL 57.600 (SEQ ID NO: 62) 15 4 CTGSQALLL57.600 (SEQ ID NO: 52) 16 208 SCTGSQALL 48.000 (SEQ ID NO: 202) 17 441NMTKLQLAL 48.000 (SEQ ID NO: 149) 18 207 DSCTGSQAL 48.000 (SEQ ID NO:61) 19 130 NAPYLPSCL 48.000 (SEQ ID NO: 144) 20 235 CMTWNQMNL 48.000(SEQ ID NO: 49)

TABLE XXXVIII Results of BIMAS HLA Peptide Binding Prediction Analysisfor Binding of Human WT1 Peptides to Mouse MHC Class I Kk Score(Estimate of Half Time of Disassociation of Start Subsequence a MoleculeContaining Rank Position Residue Listing This Subsequence) 1 81AEPHEEQCL 40.000 (SEQ ID NO: 30) 2 85 EEQCLSAFT 40.000 (SEQ ID NO: 65) 3429 DELVRHHNM 20.000 (SEQ ID NO: 53) 4 315 SETSEKRPF 20.000 (SEQ ID NO:209) 5 261 TEGQSNHST 20.000 (SEQ ID NO: 221) 6 410 SEKPFSCRW 10.000 (SEQID NO: 207) 7 272 ESDNHTTPI 10.000 (SEQ ID NO: 71) 8 318 SEKRPFMCA10.000 (SEQ ID NO: 208) 9 138 LESQPAIRN 10.000 (SEQ ID NO: 132) 10 233LECMTWNQM 10.000 (SEQ ID NO: 131) 11 298 QDVRRVPGV 10.000 (SEQ ID NO:164) 12 84 HEEQCLSAF 10.000 (SEQ ID NO: 107) 13 349 GEKPYQCDF 10.000(SEQ ID NO: 91) 14 289 HTHGVFRGI 10.000 (SEQ ID NO: 113) 15 179EDPMGQQGS 8.000 (SEQ ID NO: 64) 16 136 SCLESQPAI 5.000 (SEQ ID NO: 198)17 280 ILCGAQYRI 5.000 (SEQ ID NO: 116) 18 273 SDNHTTPIL 4.000 (SEQ IDNO: 204) 19 428 SDELVRHHN 4.000 (SEQ ID NO: 203) 20 3 SDVRDLNAL 4.000(SEQ ID NO: 206)

TABLE XXXIX Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Mouse MHC Class I Ld Score (Estimate ofHalf Time of Dis- association of a Molecule Start Containing Posi-Subsequence This Rank tion Residue Listing Subsequence) 1 163 TPSHHAAQF(SEQ ID NO: 228) 360.000 2 327 YPGCNKRYF (SEQ ID NO: 250) 300.000 3 180DPMGQQGSL (SEQ ID NO: 59) 150.000 4 26 LPVSGAAQW (SEQ ID NO: 138) 93.6005 278 TPILCGAQY (SEQ ID NO: 227) 72.000 6 141 QPAIRNQGY (SEQ ID NO: 170)60.000 7 219 TPYSSDNLY (SEQ ID NO: 231) 60.000 8 303 VPGVAPTLV (SEQ IDNO: 242) 60.000 9 120 ASSGQARMF (SEQ ID NO: 40) 50.000 10 63 PPPPPPHSF(SEQ ID NO: 158) 45.000 11 113 GPPPPSQAS (SEQ ID NO: 97) 45.000 12 157TPSYGHTPS (SEQ ID NO: 229) 39.000 13 207 DSCTGSQAL (SEQ ID NO: 61)32.500 14 110 GPFGPPPPS (SEQ ID NO: 96) 30.000 15 82 EPHEEQCLS (SEQ IDNO: 68) 30.000 16 412 KPFSCRWPS (SEQ ID NO: 123) 30.000 17 418 WPSCQKKFA(SEQ ID NO: 246) 30.000 18 221 YSSDNLYQM (SEQ ID NO: 253) 30.000 19 204TPTDSCTGS (SEQ ID NO: 230) 30.000 20 128 FPNAPYLPS (SEQ ID NO: 79)30.000

TABLE XL Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Human WT1 Peptides to Cattle HLA A20 Score (Estimate of HalfTime of Dis- association of a Molecule Start Containing Posi-Subsequence This Rank tion Residue Listing Subsequence) 1 350 EKPYQCDFK(SEQ ID NO: 66) 1000.00 2 319 EKRPFMCAY (SEQ ID NO: 67) 500.000 3 423KKFARSDEL (SEQ ID NO: 122) 500.000 4 345 RKHTGEKPY (SEQ ID NO: 184)500.000 5 390 RKFSRSDHL (SEQ ID NO: 183) 500.000 6 137 CLESQPAIR (SEQ IDNO: 47) 120.000 7 380 VKPFQCKTC (SEQ ID NO: 239) 100.000 8 407 GKTSEKPFS(SEQ ID NO: 95) 100.000 9 335 FKLSHLQMH (SEQ ID NO: 78) 100.000 10 247LKGVAAGSS (SEQ ID NO: 135) 100.000 11 370 LKRHQRRHT (SEQ ID NO: 136)100.000 12 258 VKWTEGQSN (SEQ ID NO: 240) 100.000 13 398 LKTHTRTHT (SEQID NO: 137) 100.000 14 331 NKRYFKLSH (SEQ ID NO: 145) 100.000 15 357FKDCERRFS (SEQ ID NO: 77) 100.000 16 385 CKTCQRKFS (SEQ ID NO: 46)100.000 17 294 FRGIQDVRR (SEQ ID NO: 81) 80.000 18 368 DQLKRHQRR (SEQ IDNO: 60) 80.000 19 432 VRHHNMHQR (SEQ ID NO: 243) 80.000 20 118 SQASSGQAR(SEQ ID NO: 216) 80.000

TABLE XLI Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Mouse WT1 Peptides to Human HLA A 0201 Score (Estimate ofHalf Time of Dis- association of a Molecule Start Containing Posi-Subsequence This Rank tion Residue Listing Subsequence) 1 126 RMFPNAPYL(SEQ ID NO: 293) 313.968 2 187 SLGEQQYSV (SEQ ID NO: 299) 285.163 3 10ALLPAVSSL (SEQ ID NO: 255) 181.794 4 225 NLYQMTSQL (SEQ ID NO: 284)68.360 5 292 GVFRGIQDV (SEQ ID NO: 270) 51.790 6 93 TLHFSGQFT (SEQ IDNO: 302) 40.986 7 191 QQYSVPPPV (SEQ ID NO: 290) 22.566 8 280 ILCGAQYRI(SEQ ID NO: 274) 17.736 9 441 NMTKLHVAL (SEQ ID NO: 285) 15.428 10 235CMTWNQMNL (SEQ ID NO: 258) 15.428 11 7 DLNALLPAV (SEQ ID NO: 261) 11.99812 242 NLGATLKGM (SEQ ID NO: 283) 11.426 13 227 YQMTSQLEC (SEQ ID NO:307) 8.573 14 239 NQMNLGATL (SEQ ID NO: 286) 8.014 15 309 TLVRSASET (SEQID NO: 303) 7.452 16 408 KTSEKPFSC (SEQ ID NO: 277) 5.743 17 340LQMHSRKHT (SEQ ID NO: 280) 4.752 18 228 QMTSQLECM (SEQ ID NO: 289) 4.04419 37 VLDFAPPGA (SEQ ID NO: 304) 3.378 20 302 RVSGVAPTL (SEQ ID NO: 295)1.869

TABLE XLII Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Mouse WT1 Peptides to Mouse MHC Class I Db Score (Estimate ofHalf Time of Dis- association of a Molecule Start Containing Posi-Subsequence This Rank tion Residue Listing Subsequence) 1 221 YSSDNLYQM(SEQ ID NO: 308) 312.000 2 126 RMFPNAPYL (SEQ ID NO: 293) 260.000 3 235CMTWNQMNL (SEQ ID NO: 258) 260.000 4 437 MHQRNMTKL (SEQ ID NO: 281)200.000 5 238 WNQMNLGAT (SEQ ID NO: 305) 12.000 6 130 NAPYLPSCL (SEQ IDNO: 282) 8.580 7 3 SDVRDLNAL (SEQ ID NO: 298) 7.920 8 136 SCLESQPTI (SEQID NO: 296) 7.920 9 81 AEPHEEQCL (SEQ ID NO: 254) 6.600 10 10 ALLPAVSSL(SEQ ID NO: 255) 6.600 11 218 RTPYSSDNL (SEQ ID NO: 294) 6.000 12 441NMTKLHVAL (SEQ ID NO: 285) 3.432 13 228 QMTSQLECM (SEQ ID NO: 289) 3.12014 174 HSFKHEDPM (SEQ ID NO: 272) 3.120 15 242 NLGATLKGM (SEQ ID NO:283) 2.640 16 261 TEGQSNHGI (SEQ ID NO: 301) 2.640 17 225 NLYQMTSQL (SEQID NO: 284) 2.640 18 207 DSCTGSQAL (SEQ ID NO: 263) 2.600 19 119QASSGQARM (SEQ ID NO: 288) 2.600 20 18 LGGGGGCGL (SEQ ID NO: 279) 2.600

TABLE XLIII Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Mouse WT1 Peptides to Mouse MHC Class I Kb Score (Estimate ofHalf Time of Dis- association of a Molecule Start Containing Posi-Subsequence This Rank tion Residue Listing Subsequence) 1 329 GCNKRYFKL(SEQ ID NO: 268) 24.000 2 225 NLYQMTSQL (SEQ ID NO: 284) 10.000 3 420SCQKKFARS (SEQ ID NO: 297) 3.960 4 218 RTPYSSDNL (SEQ ID NO: 294) 3.6305 437 MHQRNMTKL (SEQ ID NO: 281) 3.600 6 387 TCQRKFSRS (SEQ ID NO: 300)3.600 7 289 HTHGVFRGI (SEQ ID NO: 273) 3.000 8 130 NAPYLPSCL (SEQ ID NO:282) 3.000 9 43 PGASAYGSL (SEQ ID NO: 287) 2.400 10 155 DGAPSYGHT (SEQID NO: 260) 2.400 11 126 RMFPNAPYL (SEQ ID NO: 293) 2.200 12 128FPNAPYLPS (SEQ ID NO: 267) 2.000 13 207 DSCTGSQAL (SEQ ID NO: 263) 1.58414 3 SDVRDLNAL (SEQ ID NO: 298) 1.584 15 332 KRYFKLSHL (SEQ ID NO: 276)1.500 16 233 LECMTWNQM (SEQ ID NO: 278) 1.320 17 18 LGGGGGCGL (SEQ IDNO: 279) 1.320 18 242 NLGATLKGM (SEQ ID NO: 283) 1.200 19 123 GQARMFPN(SEQ ID NO: 269)A 1.200 20 441 NMTKLHVAL (SEQ ID NO: 285) 1.200

TABLE XLIV Results of BIMAS HLA Peptide Binding Prediction Analysis forBinding of Mouse WT1 Peptides to Mouse MHC Class I Kd Score (Estimate ofHalf Time of Dis- association of a Molecule Start Containing Posi-Subsequence This Rank tion Residue Listing Subsequence) 1 285 QYRIHTHGV(SEQ ID NO: 291) 600.000 2 424 KFARSDELV (SEQ ID NO: 275) 288.000 3 334YFKLSHLQM (SEQ ID NO: 306) 120.000 4 136 SCLESQPTI (SEQ ID NO: 296)115.200 5 239 NQMNLGATL (SEQ ID NO: 286) 115.200 6 10 ALLPAVSSL (SEQ IDNO: 255) 115.200 7 47 AYGSLGGPA (SEQ ID NO: 256) 86.400 8 180 DPMGQQGSL(SEQ ID NO: 262) 80.000 9 270 GYESDNHTA (SEQ ID NO: 271) 72.000 10 192QYSVPPPVY (SEQ ID NO: 292) 60.000 11 326 AYPGCNKRY (SEQ ID NO: 257)60.000 12 289 HTHGVFRGI (SEQ ID NO: 273) 57.600 13 4 DVRDLNALL (SEQ IDNO: 264) 57.600 14 126 RMFPNAPYL (SEQ ID NO: 293) 57.600 15 209CTGSQALLL (SEQ ID NO: 259) 48.000 16 86 EQCLSAFTL (SEQ ID NO: 265)48.000 17 302 RVSGVAPTL (SEQ ID NO: 295) 48.000 18 218 RTPYSSDNL (SEQ IDNO: 294) 48.000 19 272 ESDNHTAPI (SEQ ID NO: 266) 48.000 20 225NLYQMTSQL (SEQ ID NO: 284) 48.000

TABLE XLV Results of TSites Peptide Binding Prediction Analysis forHuman WT1 Peptides Capable of Eliciting a Helper T cell Response PeptideSequence p6-23 RDLNALLPAVPSLGGGG (SEQ ID NO: 1) p30-35 GAAQWA (SEQ IDNO: 309) p45-56 ASAYGSLGGPAP (SEQ ID NO: 310) p91-105 AFTVHFSGQFTGTAG(SEQ ID NO: 311) p117-139 PSQASSGQARMFPNAPYLPSCLE (SEQ ID NO: 2)p167-171 HAAQF (SEQ ID NO: 312) p202-233CHTPTDSCTGSQALLLRTPYSSDNLYQMTSQL (SEQ ID NO: 313) p244-262GATLKGVAAGSSSSVKWTE (SEQ ID NO: 4) p287-318RIHTHGVFRGIQDVRRVPGVAPTLVRSASETS (SEQ ID NO: 314) p333-336 RYFK (SEQ IDNO: 315) p361-374 ERRFSRSDQLKRHQ (SEQ ID NO: 316) p389-410QRKFSRSDHLKTHTRTHTGKTS (SEQ ID NO: 317) p421-441 CQKKFARSDELVRHHNMHQRN(SEQ ID NO: 318)

Certain CTL peptides (shown in Table XLVI) were selected for furtherstudy. For each peptide in Table XLVI, scores obtained using BIMAS HLApeptide binding prediction analysis are provided.

TABLE XLVI WT1 Peptide Sequences and HLA Peptide Binding PredictionsPeptide Sequence Comments p329-337 GCNKRYFKL Score 24,000 (SEQ ID NOs:90 and 268) p225-233 NLYQMTSQL binds also to class II (SEQ ID NOs: 147and HLA A2, Kd, and 284) score 10,000 p235-243 CMTWNQMNL binds also toHLA A2, (SEQ ID NOs: 49 and score 5,255,712 258) p126-134 RMFPNAPYLbinds also to Kd, (SEQ ID NOs: 185 class II and HLA A2, and 293) score1,990,800 p221-229 YSSDNLYQM binds also to Ld, (SEQ ID NOs: 253 score312,000 and 308) p228-236 QMTSQLECM score 3,120 (SEQ ID NOs: 169 and289) p239-247 NQMNLGATL binds also to HLA (SEQ ID NOs: 151 A 0201, Kd,score and 286) 8,015 mouse SCLESQPTI binds also to Kd, p136-144 (SEQ IDNO: 296) 1 mismatch to human human SCLESQPAI score 7,920 p136-144 (SEQID NO: 198) mouse ALLPAVSSL binds also to Kd, p10-18 (SEQ ID NO: 255)HLA A2, 1 mismatch to human human ALLPAVPSL score 6,600 p10-18 (SEQ IDNO: 34)

Peptide binding to C57BI/6 murine MHC was confirmed using the leukemiacell line RMA-S, as described by Ljunggren et al., Nature 346:476-480,1990. In brief, RMA-S cells were cultured for 7 hours at 26° C. incomplete medium supplemented with 1% FCS. A total of 10⁶ RMA-S cellswere added into each well of a 24-well plate and incubated either aloneor with the designated peptide (25 ug/ml) for 16 hours at 26° C. andadditional 3 hours at 37° C. in complete medium. Cells were then washedthree times and stained with fluorescein isothiocyanate-conjugated antiD^(b) or anti-K^(b) antibody (PharMingen, San Diego, Calif.). Labeledcells were washed twice, resuspended and fixed in 500 ul of PBS with 1%paraformaldehyde and analyzed for fluorescence intensity in a flowcytometer (Becton-Dickinson FACSCALIBUR™). The percentage of increase ofD^(b) or K^(b) molecules on the surface of the RMA-S cells was measuredby increased mean fluorescent intensity of cells incubated with peptidecompared with that of cells incubated in medium alone.

Mice were immunized with the peptides capable of binding to murine classI MHC. Following immunization, spleen cells were stimulated in vitro andtested for the ability to lyse targets incubated with WT1 peptides. CTLwere evaluated with a standard chromium release assay (Chen et al.,Cancer Res. 54:1065-1070, 1994). 10⁶ target cells were incubated at 37°C. with 150 μCi of sodium ⁵¹Cr for 90 minutes, in the presence orabsence of specific peptides. Cells were washed three times andresuspended in RPMI with 5% fetal bovine serum. For the assay, 10⁴⁵¹Cr-labeled target cells were incubated with different concentrationsof effector cells in a final volume of 200 μl in U-bottomed 96-wellplates. Supernatants were removed after 4 to 7 hours at 37° C., and thepercentage specific lysis was determined by the formula:

% specific lysis=100×(experimental release−spontaneous release)/(maximumrelease−spontaneous release).

The results, presented in Table XLVII, show that some WT1 peptides canbind to class I MHC molecules, which is essential for generating CTL.Moreover, several of the peptides were able to elicit peptide specificCTL (FIGS. 9A and 9B), as determined using chromium release assays.Following immunization to CTL peptides p10-18 human, p136-144 human,p136-144 mouse and p235-243, peptide specific CTL lines were generatedand clones were established. These results indicate that peptidespecific CTL can kill malignant cells expressing WT1.

TABLE XLVII Binding of WT1 CTL Peptides to mouse B6 class I antigensPeptide Binding Affinity to Mouse MHC Class I Positive control  91%negative control 0.5.-1.3% p235-243 33.6%  p136-144 mouse 27.9% p136-144 human  52% p10-18: human 2.2% p225-233 5.8% p329-337 1.2%p126-134 0.9% p221-229 0.8% p228-236 1.2% p239-247   1%

Example 5 Use of A WT1 Polypeptide to Elicit WT1 Specific CTL in Mice

This Example illustrates the ability of a representative WT1 polypeptideto elicit CTL immunity capable of killing WT1 positive tumor cell lines.

P117-139, a peptide with motifs appropriate for binding to class I andclass II MHC, was identified as described above using TSITES and BIMASHLA peptide binding prediction analyses. Mice were immunized asdescribed in Example 3. Following immunization, spleen cells werestimulated in vitro and tested for the ability to lyse targets incubatedwith WT1 peptides, as well as WT1 positive and negative tumor cells. CTLwere evaluated with a standard chromium release assay. The results,presented in FIGS. 10A-10D, show that P117 can elicit WT1 specific CTLcapable of killing WT1 positive tumor cells, whereas no killing of WT1negative cells was observed. These results demonstrate that peptidespecific CTL in fact kill malignant cells expressing WT1 and thatvaccine and T cell therapy are effective against malignancies thatexpress WT1.

Similar immunizations were performed using the 9-mer class I MHC bindingpeptides p136-144, p225-233, p235-243 as well as the 23-mer peptidep117-139. Following immunization, spleen cells were stimulated in vitrowith each of the 4 peptides and tested for ability to lyse targetsincubated with WT1 peptides. CTL were generated specific for p136-144,p235-243 and p117-139, but not for p225-233. CTL data for p235-243 andp117-139 are presented in FIGS. 11A and 11B. Data for peptides p136-144and p225-233 are not depicted.

CTL lysis demands that the target WT1 peptides are endogenouslyprocessed and presented in association with tumor cell class I MHCmolecules. The above WT1 peptide specific CTL were tested for ability tolyse WT1 positive versus negative tumor cell lines. CTL specific forp235-243 lysed targets incubated with the p235-243 peptides, but failedto lyse cell lines that expressed WT1 proteins (FIG. 11A). By markedcontrast, CTL specific for p117-139 lysed targets incubated withp117-139 peptides and also lysed malignant cells expressing WT1 (FIG.11B). As a negative control, CTL specific for p117-139 did not lyse WT1negative EL-4 (also referred to herein as E10).

Specificity of WT1 specific lysis was confirmed by cold targetinhibition (FIGS. 12A-12B). Effector cells were plated for variouseffector: target ratios in 96-well U-bottom plates. A ten-fold excess(compared to hot target) of the indicated peptide-coated target without⁵¹Cr labeling was added. Finally, 10⁴ ⁵¹Cr-labeled target cells per wellwere added and the plates incubated at 37° C. for 4 hours. The totalvolume per well was 200 μl.

Lysis of TRAMP-C by p117-139 specific CTL was blocked from 58% to 36% byEL-4 incubated with the relevant peptide p117-139, but not with EL-4incubated with an irrelevant peptide (FIG. 12A). Similarly, lysis ofBLK-SV40 was blocked from 18% to 0% by EL-4 incubated with the relevantpeptide p117-139 (FIG. 12B). Results validate that WT1 peptide specificCTL specifically kill malignant cells by recognition of processed WT1.

Several segments with putative CTL motifs are contained within p117-139.To determine the precise sequence of the CTL epitope all potential 9-merpeptides within p117-139 were synthesized (Table XLVIII). Two of thesepeptides (p126-134 and p130-138) were shown to bind to H-2^(b) class Imolecules (Table XLVIII). CTL generated by immunization with p117-139lysed targets incubated with p126-134 and p130-138, but not the other9-mer peptides within p117-139 (FIG. 13A).

The p117-139 specific CTL line was restimulated with either p126-134 orp130-138. Following restimulation with p126-134 or p130-138, both T celllines demonstrated peptide specific lysis, but only p130-138 specificCTL showed lysis of a WT1 positive tumor cell line (FIGS. 13B and 13C).Thus, p130-138 appears to be the naturally processed epitope.

TABLE XLVIII Binding of WT1 CTL 9mer Peptides within p117-139 to mouseB6 class I antigens Binding Affinity to Mouse MHC Peptide Class IP117-125 PSQASSGQA (SEQ ID NO: 221) 2% P118-126 SQASSGQAR (SEQ ID NO:216) 2% P119-127 QASSGQARM (SEQ ID Nos: 161 and 2% 288) P120-128ASSGQARMF (SEQ ID NO: 40 1% P121-129 SSGQARMFP (SEQ ID NO: 222) 1%P122-130 SGQARMFPN (SEQ ID NO: 212) 1% P123-131 GQARMFPNA (SEQ ID Nos:98 and 1% 269) P124-132 QARMFPNAP (SEQ ID NO: 223) 1% P125-133 ARMFPNAPY(SEQ ID NO: 38) 1% P126-134 RMFPNAPYL (SEQ ID NOs: 185 and 79%  293)P127-135 MFPNAPYLP (SEQ ID NO: 224) 2% P128-136 FPNAPYLPS (SEQ ID NOs:79 and 267) 1% P129-137 PNAPYLPSC (SEQ ID NO: 225) 1% P130-138 NAPYLPSCL(SEQ ID NOs: 144 and 79%  282) P131-139 APYLPSCLE (SEQ ID NO: 226) 1%

Example 6 Identification of WT1 Specific mRNA in Mouse Tumor Cell Lines

This Example illustrates the use of RT-PCR to detect WT1 specific mRNAin cells and cell lines.

Mononuclear cells were isolated by density gradient centrifugation, andwere immediately frozen and stored at −80° C. until analyzed by RT-PCRfor the presence of WT1 specific mRNA. RT-PCR was generally performed asdescribed by Fraizer et al., Blood 86:4704-4706, 1995. Total RNA wasextracted from 10⁷ cells according to standard procedures. RNA pelletswere resuspended in 25 μL diethylpyrocarbonate treated water and useddirectly for reverse transcription. The zinc-finger region (exons 7 to10) was amplified by PCR as a 330 bp mouse cDNA. Amplification wasperformed in a thermocycler during one or, when necessary, twosequential rounds of PCR. AMPLITAQ® DNA Polymerase (Perkin Elmer Cetus,Norwalk, Conn.), 2.5 mM MgCl₂ and 20 μmol of each primer in a totalreaction volume of 50 μl were used. Twenty μL aliquots of the PCRproducts were electrophoresed on 2% agarose gels stained with ethidiumbromide. The gels were photographed with POLAROID® film (Polaroid 667,Polaroid Ltd., Hertfordshire, England). Precautions against crosscontamination were taken following the recommendations of Kwok andHiguchi, Nature 339:237-238, 1989. Negative controls included the cDNA-and PCR-reagent mixes with water instead of cDNA in each experiment. Toavoid false negatives, the presence of intact RNA and adequate cDNAgeneration was evaluated for each sample by a control PCR using β-actinprimers. Samples that did not amplify with these primers were excludedfrom analysis.

Primers for amplification of WT1 in mouse cell lines were: P115:1458-1478: 5′CCC AGG CTG CM TAA GAG ATA 3′ (forward primer; SEQ IDNO:21); and P116: 1767-1787: 5′ ATG TTG TGA TGG CGG ACC AAT 3′ (reverseprimer; SEQ ID NO:22) (see Inoue et al, Blood 88:2267-2278, 1996;Fraizer et al., Blood 86:4704-4706, 1995).

Beta Actin primers used in the control reactions were: 5′ GTG GGG CGCCCC AGG CAC CA 3′ (sense primer; SEQ ID NO:23); and 5′ GTC CTT MT GTCACG CAC GAT TTC 3′ (antisense primer; SEQ ID NO:24)

Primers for use in amplifying human WT1 include: P117: 954-974: 5′ GGCATC TGA GAC CAG TGA GAA 3′ (SEQ ID NO:25); and P118: 1434-1414: 5′ GAGAGT CAG ACT TGA AAG CAGT 3′ (SEQ ID NO:5). For nested RT-PCR, primersmay be: P119: 1023-1043: 5′ GCT GTC CCA CTT ACA GAT GCA 3′ (SEQ IDNO:26); and P120: 1345-1365: 5′ TCA MG CGC CAG CTG GAG TTT 3′ (SEQ IDNO:27).

Table XLVIII shows the results of WT1 PCR analysis of mouse tumor celllines. Within Table IV, (+++) indicates a strong WT1 PCR amplificationproduct in the first step RT PCR, (++) indicates a WT1 amplificationproduct that is detectable by first step WT1 RT PCR, (+) indicates aproduct that is detectable only in the second step of WT1 RT PCR, and(−) indicates WT1 PCR negative.

TABLE XLIX Detection of WT1 mRNA in Mouse Tumor Cell Lines Cell Line WT1mRNA K562 (human leukemia; ATCC): Positive control; (Lozzio +++ andLozzio, Blood 45: 321-334, 1975) TRAMPC (SV40 transformed prostate, B6);Foster et al., +++ Cancer Res. 57: 3325-3330, 1997 BLK-SV40 HD2(SV40-transf. fibroblast, B6; ATCC); ++ Nature 276: 510-511, 1978 CTLL(T-cell, B6; ATCC); Gillis, Nature 268: 154-156, + 1977) FM (FBL-3subline, leukemia, B6); Glynn and Fefer, + Cancer Res. 28: 434-439, 1968BALB 3T3 (ATCC); Aaroston and Todaro, J. Cell. + Physiol. 72: 141-148,1968 S49.1 (Lymphoma, T-cell like, B/C; ATCC); Horibata and + Harris,Exp. Cell. Res. 60: 61, 1970 BNL CL.2 (embryonic liver, B/C; ATCC);Nature + 276: 510-511, 1978 MethA (sarcoma, B/C); Old et al., Ann. NYAcad. Sci. − 101: 80-106, 1962 P3.6.2.8.1 (myeloma, B/C; ATCC); Proc.Natl. Acad. Sci. − USA 66: 344, 1970 P2N (leukemia, DBA/2; ATCC);Melling et al., J. − Immunol. 117: 1267-1274, 1976 BCL1 (lymphoma, B/C;ATCC); Slavin and Strober, − Nature 272: 624-626, 1977 LSTRA (lymphoma,B/C); Glynn et al., Cancer Res. − 28: 434-439, 1968 E10/EL-4 (lymphoma,B6); Glynn et al., Cancer Res. − 28: 434-439, 1968

Example 7 Expression in E. Coli of WT1 TRX Fusion Construct

The truncated open reading frame of WT1 (WT1B) was PCR amplified withthe following primers:

Forward Primer starting at amino acid 2 P-37 (SEQ ID NO. 347)5′ ggctccgacgtgcgggacctg 3′ Tm 64° C. Reverse Primer creating EcoRI siteafter stop codon P-23 (SEQ ID NO. 348) 5′ gaattctcaaagcgccagctggagtttggt3′ Tm 63° C.

The PCR was performed under the following conditions:

10 μl 10X Pfu buffer 1 μl 10 mM dNTPs 2 μl 10 μM each oligo 83 μLsterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, CA) 50 ngDNA (pPDM FL WT1) 96° C. 2 minutes 96° C. 20 seconds 63° C. 15 seconds72° C. 3 minutes × 40 cycles 72° C. 4 minutes

The PCR product was digested with EcoRI restriction enzyme, gel purifiedand then cloned into pTrx 2H vector (a modified pET28 vector with a Trxfusion on the N-terminal and two His tags surrounding the Trx fusion.After the Trx fusion there exists protease cleavage sites for thrombinand enterokinase). The pTrx2H construct was digested with StuI and EcoRIrestriction enzymes. The correct constructs were confirmed by DNAsequence analysis and then transformed into BL21 (DE3) pLys S and BL21(DE3) CodonPlus expression host cells.

Example 8 Expression in E. Coli of WT1 A His Tag Fusion Constructs

The N-terminal open reading frame of WT1 (WT1A) was PCR amplified withthe following primers:

Forward Primer starting at amino acid 2 P-37 (SEQ ID NO. 347)5′ggctccgacgtgcgggacctg 3′ Tm 64° C. Reverse Primer creating EcoRI siteafter an artificial stop codon put after amino acid 249. PDM-335 (SEQ IDNO. 348) 5′gaattctcaaagcgccagctggagtttggt 3′ Tm 64° C.

The PCR was performed under the following conditions:

10 μl 10X Pfu buffer 1 μl 10 mM dNTPs 2 μl 10 μM each oligo 83 μLsterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, CA) 50 ngDNA (pPDM FL WT1) 96° C. 2 minutes 96° C. 20 seconds 63° C. 15 seconds72° C. 1 minute 20 seconds × 40 cycles 72° C. 4 minutes

The PCR product was digested with EcoRI restriction enzyme, gel purifiedand then cloned into pPDM, a modified pET28 vector with a His tag inframe, which had been digested with Eco72I and EcoRI restrictionenzymes. The PCR product was also transformed into pTrx 2H vector. ThepTrx2H construct was digested with StuI and EcoRI restriction enzymes.The correct constructs were confirmed by DNA sequence analysis and thentransformed into BL21 (DE3) pLys S and BL21 (DE3) CodonPlus expressionhost cells.

Example 9 Expression in E. Coli of WT1 B His Tag Fusion Constructs

The truncated open reading frame of WT1 (WT1A) was PCR amplified withthe following primers:

Forward Primer starting at amino acid 250 PDM-346 (SEQ ID NO. 351)5′ cacagcacagggtacgagagc 3′ Tm 58° C. Reverse Primer creating EcoRI siteafter stop codon P-23 (SEQ ID NO. 348) 5′gaattctcaaagcgccagctggagtttggt3′ Tm 63° C.

The PCR was performed under the following conditions:

10 μl 10X Pfu buffer 1 μl 10 mM dNTPs 2 μl 10 μM each oligo 83 μLsterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, CA) 50 ngDNA (pPDM FL WT1) 96° C. 2 minutes 96° C. 20 seconds 63° C. 15 seconds72° C. 1 minute 30 seconds × 40 cycles 72° C. 4 minutes

The PCR product was digested with EcoRI restriction enzyme, gel purifiedand then cloned into pPDM, a modified pET28 vector with a His tag inframe, which had been digested with Eco72I and EcoRI restrictionenzymes. The PCR product was also transformed into pTrx 2H vector. ThepTrx 2H construct was digested with StuI and EcoRI restriction enzymes.The correct constructs were confirmed by DNA sequence analysis and thentransformed into BL21 (DE3) pLys S and BL21 (DE3) CodonPlus expressionhost cells.

For Examples 7-9, the following SEQ ID NOs. are disclosed:

SEQ ID NO. 327 is the determined cDNA sequence for Trx_WT1_BSEQ ID NO. 328 is the determined cDNA sequence for Trx_WT1_ASEQ ID NO. 329 is the determined cDNA sequence for Trx_WT1SEQ ID NO. 330 is the determined cDNA sequence for WT1_ASEQ ID NO. 331 is the determined cDNA sequence for WT1_BSEQ ID NO. 332 is the predicted amino acid sequence encoded by SEQ IDNo.SEQ ID NO. 333 is the predicted amino acid sequence encoded by SEQ IDNo. 328SEQ ID NO. 334 is the predicted amino acid sequence encoded by SEQ IDNo. 329SEQ ID NO. 335 is the predicted amino acid sequence encoded by SEQ IDNo. 330SEQ ID NO. 336 is the predicted amino acid sequence encoded by SEQ IDNo. 331

Example 10 Truncated Forms of WT1 Expressed in E. Coli

Three reading frames of WT1 were amplified by PCR using the followingprimers:

For WT1 Tr2: PDM-441 (SEQ ID NO. 353) 5′ cacgaagaacagtgcctgagcgcattcac3′ Tm 63° C. PDM-442 (SEQ ID NO. 354)5′ ccggcgaattcatcagtataaattgtcactgc 3′ TM 62° C. For WT1 Tr3: PDM-443(SEQ ID NO. 355) 5′ caggctttgctgctgaggacgccc 3′ Tm 64° C. PDM-444 (SEQID NO. 356) 5′ cacggagaattcatcactggtatggtttctcacc Tm 64° C. For WT1 Tr4:PDM-445 (SEQ ID NO. 357) 5′ cacagcaggaagcacactggtgagaaac 3′ Tm 63° C.PDM-446 (SEQ ID NO. 358) 5′ ggatatctgcagaattctcaaagcgccagc 3′ TM 63° C.

The PCR was performed under the following conditions:

10 μl 10X Pfu buffer 1 μl 10 mM dNTPs 2 μl 10 μM each oligo 83 μLsterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, CA) 50 ngDNA (pPDM FL WT1) 96° C. 2 minutes 96° C. 20 seconds 63° C. 15 seconds72° C. 30 seconds × 40 cycles 72° C. 4 minutes

The PCR products were digested with EcoRI and cloned into pPDM His (amodified pET28 vector with a His tag in frame on the 5′ end) which hasbeen digested with Eco72I and EcoRI. The constructs were confirmed to becorrect through sequence analysis and transformed into BL21 pLys S andBL21-CODONPLUS® cells or BLR pLys S and BL21-CODONPLUS® cells.

Example 11 WT1 C (Amino Acids 76-437) and WT1 D (Amino Acids 91-437)Expression in E. Coli

The WT1 C reading frame was amplified by PCR using the followingprimers:

PDM-504 (SEQ ID NO. 359) 5′ cactccttcatcaaacaggaac 3′ Tm 61° C. PDM-446(SEQ ID NO. 358) 5′ ggatatctgcagaattctcaaagcgccagc 3′ Tm 63° C.

The PCR was performed under the following conditions:

10 μl 10X Pfu buffer 1 μl 10 mM dNTPs 2 μl 10 μM each oligo 83 μLsterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, CA) 50 ngDNA (pPDM FL WT1) 96° C. 2 minutes 96° C. 20 seconds 63° C. 15 seconds72° C. 2 minutes × 40 cycles 72° C. 4 minutes

The PCR product was digested with EcoRI and cloned into pPDM His whichhad been digested with Eco72I and EcoRI. The sequence was confirmedthrough sequence analysis and then transformed into BLR pLys S and BLRwhich is co-transformed with CODONPLUS® RP.

Example 12 Synthetic Production of WT1 TR-1 by Annealing OverlappingOligos

This example was performed to determine the effect of changing prolinecodon usage on expression.

The following pairs of oligos were annealed:

1. PDM-505 (SEQ ID NO. 361) 5′ ggttccgacgtgcgggacctgaacgcactgctg 3′PDM-506 (SEQ ID NO. 362) 5′ ctgccggcagcagtgcgttcaggtcccgcacgtcggaacc 3′2. PDM-507 (SEQ ID NO. 363) 5′ ccggcagttccatccctgggtggcggtggaggctg 3′PDM-508 (SEQ ID NO. 364) 5′ cggcagtgcgcagcctccaccgccacccagggatggaa 3′ 3.PDM-509 (SEQ ID NO. 365) 5′ cgcactgccggttagcggtgcagcacagtgggctc 3′PDM-510 (SEQ ID NO. 366) 5′ cagaactggagcccactgtgctgcaccgctaac 3′ 4.PDM-511 (SEQ ID NO. 367) 5′ cagttctggacttcgcaccgcctggtgcatccgcatac 3′PDM-512 (SEQ ID NO. 368) 5′ cagggaaccgtatgcggatgcaccaggcggtgcgaagtc 3′5. PDM-513 (SEQ ID NO. 369) 5′ ggttccctgggtggtccagcacctccgcccgcaacgcc 3′PDM-514 (SEQ ID NO. 370) 5′ ggcggtgggggcgttgcgggcggaggtgctggaccacc 3′ 6.PDM-515 (SEQ ID NO. 371) 5′ cccaccgcctccaccgcccccgcactccttcatcaaacag 3′PDM-516 (SEQ ID NO. 372) 5′ ctaggttcctgtttgatgaaggagtgcgggggcggtgga 3′7. PDM-517 (SEQ ID NO. 373) 5′ gaacctagctggggtggtgcagaaccgcacgaagaaca 3′PDM-518 (SEQ ID NO. 374) 5′ ctcaggcactgttcttcgtgcggttctgcaccaccccag 3′8. PDM-519 (SEQ ID NO. 375) 5′ gtgcctgagcgcattctgagaattctgcagat 3′PDM-520 (SEQ ID NO. 376) 5′ gtgtgatggatatctgcagaattctcagaatgcg 3′

Each oligo pair was separately combined then annealed. The pairs werethen ligated together and one μl of ligation mix was used for PCRconditions below:

10 μl 10X Pfu buffer 1 μl 10 mM dNTPs 2 μl 10 μM each oligo 83 μLsterile water 1.5 μl Pfu DNA polymerase (Stratagene, La Jolla, CA) 96°C. 2 minutes 96° C. 20 seconds 63° C. 15 seconds 72° C. 30 seconds × 40cycles 72° C. 4 minutes

The PCR product was digested with EcoRI and cloned into pPDM His whichhad been digested with Eco72I and EcoRI. The sequence was confirmed andthen transformed into BLR pLys S and BLR which is co-transformed withCODONPLUS® RP.

For examples 10-12, the following SEQ ID NOs. are disclosed:

SEQ ID NO:337 is the determined cDNA sequence for WT1_Tr1SEQ ID NO:338 is the determined cDNA sequence for WT1_Tr2SEQ ID NO:339 is the determined cDNA sequence for WT1_Tr3SEQ ID NO:340 is the determined cDNA sequence for WT1_Tr4SEQ ID NO:341 is the determined cDNA sequence for WT1_CSEQ ID NO:342 is the predicted amino acid sequence encoded by SEQ IDNO:337SEQ ID NO:343 is the predicted amino acid sequence encoded by SEQ IDNO:338SEQ ID NO:344 is the predicted amino acid sequence encoded by SEQ IDNO:339SEQ ID NO:345 is the predicted amino acid sequence encoded by SEQ IDNO:340SEQ ID NO:346 is the predicted amino acid sequence encoded by SEQ IDNO:341

The WT1 C sequence represents a polynucleotide having the coding regionsof TR2, TR3 and TR4.

The WT1 TR-1 synthetic sequence represents a polynucleotide in whichalternative codons for proline were substituted for the native codons,producing a polynucleotide capable of expressing WT1 TR-1 in E. coli.

Example 13 Evaluation of the Systemic Histopathological andToxicological Effects of WT1 Immunization in Mice

The purpose of this example is to analyze the immunogenicity andpotential systemic histopathological and toxicological effects of WT1protein immunization in a multiple dose titration in mice.

The experimental design for immunization of mice with WT1 protein isoutlined in Table L.

TABLE L Experimental Design of WT1 Immunization in Mice His- tologyCorixa Dose Total No. Group Group Treatment Description Level (Females)1 0 No treatment 0 4 2 1.1 MPL ®-SE (adjuvants alone), 10 ug 4 6x, 1week apart 3 1.2 MPL ®-SE, 3x, 2 weeks apart 10 ug 4 4 2.1 Ra12-WT1 +MPL ®-SE, 6x 25 ug 4 5 2.2 Ra12-WT1 + MPL ®-SE, 3x 25 ug 4 6 3.1Ra12-WT1 + MPL ®-SE, 6x 100 ug 4 7 3.2 Ra12-WT1 + MPL ®-SE, 3x 100 ug 48 4.1 Ra12-WT1 + MPL ®-SE, 6x 1000 ug 4 9 4.2 Ra12-WT1 + MPL ®-SE, 3x1000 ug 4 Vaccination to WT1 protein using MPL ®-SE as adjuvant, in amultiple dose titration study (doses ranging from 25 μg, 100 μg to 1000μg WT1 protein) in female C57/B6 mice elicited a strong WT1-specificantibody response (FIG. 19) and cellular T-cell responses (FIG. 20).

No systemic histopathological or toxicological effects of immunizationwith WT1 protein was observed. No histological evidence for toxicity wasseen in the following tissues: adrenal gland, brain, cecum, colon,duodenum, eye, femur and marrow, gall bladder, heart, ileum, jejunum,kidney, larynx, lacrimal gland, liver, lung, lymph node, muscle,esophagus, ovary, pancreas, parathyroid, salivary gland, sternum andmarrow, spleen, stomach, thymus, trachea, thyroid, urinary bladder anduterus.

Special emphasis was put on evaluation of potential hematopoietictoxicity. The myeloid/erythroid ratio in sternum and femur marrow wasnormal. All evaluable blood cell counts and blood chemistry (BUN,creatinine, bilirubin, albumin, globulin) were within the normal range(Table LI).

Given that existent immunity to WT1 is present in some patients withleukemia and that vaccination to WT1 protein can elicit WT1 specific Aband cellular T-cell responses in mice without toxicity to normaltissues, these experiments validate WT1 as a tumor/leukemia vaccine.

TABLE LI Clinical Chemistry and Hematology Analysis Table LI: WT1 DoseTitration Study Clinical Chemistry and Hematology Analysis K/uL M/uLg/dl % fL pg % WBC RBC Hg. HCT MCV MCH MCHC Normal Animal # 5.4-16.06.7-12.5 10.2-16.6 32-54 31-62 9.2-20.8 22.0-35.5 Group 1  1 (0) 5.68.41 12.8 43.5 53 15.2 29.4  2 (0) 5.5 9.12 13.4 47.5 53 14.7 28.2  3(0) 7.5 9.22 13.5 48 54 14.7 28.1  4 (0) 3.9 9.27 13.6 46 52 14.7 29.6Mean 5.6 9.0 13.3 46.3 53.0 14.8 28.8 STD 1.5 0.4 0.4 2.0 0.8 0.3 0.8Group 2  5 (1.5) 6.6 9 13.1 46 54 14.5 28.5  6 (1.6) 5.2 8.58 12.6 44 5314.7 28.6  7 (1.7) 7.8 9.21 13.6 46 53 14.7 29.6  8 (1.8) 6.3 NA NA 41NA NA NA Mean 6.5 8.9 13.1 44.3 53.3 14.6 28.9 STD 1.1 0.3 0.5 2.4 0.60.1 0.6 Group 3  9 (2.5) 8.3 9.16 13.6 50.3 55 14.9 27.1 10 (2.6) 5 8.7813 44.2 50 14.8 29.3 11 (2.7) 4 8.94 13.2 48.3 54 14.7 27.3 12 (2.8) 8.2NA NA 41 NA NA NA Mean 6.4 9.0 13.3 46.0 53.0 14.8 27.9 STD 2.2 0.2 0.34.2 2.6 0.1 1.2 Group 4 13 (3.5) 6.1 8.82 13.1 46 54 14.9 28.5 14 (3.6)6.1 8.64 12.9 46 54 15 28 15 (3.7) 9.3 8.93 13.2 48 55 14.8 27.5 16(3.8) 4.8 8.19 12.6 44 55 15.3 28.6 Mean 6.6 8.6 13.0 46.0 54.5 15.028.2 STD 1.9 0.3 0.3 1.6 0.6 0.2 0.5 Group 5 17 (4.5) 3.1 8.48 12.6 4654 14.9 27.5 18 (4.6) 5.7 9.12 13.7 48 54 15 28.5 19 (4.7) 5.3 8.58 1344.5 55 15.2 29.2 20 (4.8) 5.3 NA NA 40 NA NA NA Mean 4.9 8.7 13.1 44.654.3 15.0 28.4 STD 1.2 0.3 0.6 3.4 0.6 0.2 0.9 Group 6 21 (1.1) 3.5 9.3613.5 37.6 40 14.4 35.9 22 (1.2) 6.9 8.93 13.6 37.3 42 15.3 36.6 23 (1.3)3.6 8.3 12.5 35.3 43 15.1 35.5 24 (1.4) NA NA NA NA NA NA NA Mean 4.78.9 13.2 36.7 41.7 14.9 36.0 STD 1.9 0.5 0.6 1.3 1.5 0.5 0.6 Group 7 25(2.1) 4 NA NA 40 NA NA NA 26 (2.2) 7.4 9.12 13.2 38.5 42 14.5 34.3 27(2.3) 4.5 8.19 12.1 34.5 42 14.8 35.1 28 (2.4) 5.8 8.25 12.3 34.1 4114.9 36.1 Mean 5.4 8.5 12.5 36.8 41.7 14.7 35.2 STD 1.5 0.5 0.6 2.9 0.60.2 0.9 Group 8 29 (3.1) 5.1 8.53 12.6 34.9 41 14.7 36 30 (3.2) 7.6 8.4213 36.1 43 15.4 35.9 31 (3.3) 3.4 8.45 12.6 34.9 41 14.9 36.1 32 (3.4)6.1 8.11 12.3 34.8 43 15.2 35.5 Mean 5.6 8.4 12.6 35.2 42.0 15.1 35.9STD 1.8 0.2 0.3 0.6 1.2 0.3 0.3 Group 9 33 (4.1) NA NA NA NA NA NA NA 34(4.2) 4.5 8.63 12.8 36.2 42 14.8 35.2 35 (4.3) 3.9 8.85 13 36.6 41 14.735.6 36 (4.4) 4.7 8.14 12.3 33.8 42 15.1 36.3 Mean 4.4 8.5 12.7 35.541.7 14.9 35.7 STD 0.4 0.4 0.4 1.5 0.6 0.2 0.6 yes/no K/uL Abs. Abs.Abs. Abs. Abs. Abs. Plt. clump Platelets Baso Eos Bands Polys Lymph MonoNormal 0.0-0.15 0.0-0.51 0.0-0.32 8.0-42.9 8.0-18.0 0.0-1.5 Animal # no150-1500 K/uL K/uL K/uL K/uL K/uL K/uL Group 1  1 (0) yes 726 0 56 0 3365208 0  2 (0) no 860 0 0 0 55 5445 0  3 (0) no 875 0 375 0 525 6525 75 4 (0) yes 902 0 0 0 156 3744 0 Mean 840.8 0.0 107.8 0.0 268.0 5230.518.8 STD 78.4 0.0 180.1 0.0 207.0 1144.8 37.5 Group 2  5 (1.5) no 1193 0132 0 792 5214 462  6 (1.6) no 1166 0 52 0 624 4472 52  7 (1.7) no 10870 234 0 1170 6396 0  8 (1.8) yes NA 0 126 0 126 5922 126 Mean 1148.7 0.0136.0 0.0 678.0 5501.0 160.0 STD 55.1 0.0 74.8 0.0 433.1 840.5 207.9Group 3  9 (2.5) no 705 0 166 0 664 7387 83 10 (2.6) no 1140 0 150 0 5004350 0 11 (2.7) no 952 0 120 0 680 3200 0 12 (2.8) yes NA 0 164 0 6567216 164 Mean 932.3 0.0 150.0 0.0 625.0 5538.3 61.8 STD 218.2 0.0 21.20.0 83.9 2090.6 78.6 Group 4 13 (3.5) no 785 0 488 0 732 4636 244 14(3.6) yes 973 0 0 0 488 5307 305 15 (3.7) yes 939 0 465 0 558 7812 46516 (3.8) yes 1622 0 192 0 480 4080 48 Mean 1079.8 0.0 286.3 0.0 564.55458.8 265.5 STD 370.6 0.0 233.4 0.0 117.0 1647.1 172.4 Group 5 17 (4.5)no 892 0 31 0 620 2449 0 18 (4.6) yes 966 57 114 0 855 4674 0 19 (4.7)yes 883 0 53 0 742 4452 53 20 (4.8) yes NA 0 106 0 53 5141 0 Mean 913.714.3 76.0 0.0 567.5 4179.0 13.3 STD 45.5 28.5 40.4 0.0 356.2 1188.5 26.5Group 6 21 (1.1) yes 784 0 35 0 385 2870 210 22 (1.2) yes 806 0 69 0 2076486 138 23 (1.3) yes 790 0 180 0 396 2988 36 24 (1.4) NA NA NA NA NA NANA NA Mean 793.3 0.0 94.7 0.0 329.3 4114.7 128.0 STD 11.4 0.0 75.8 0.0106.1 2054.5 87.4 Group 7 25 (2.1) yes NA 0 80 0 200 3720 0 26 (2.2) yes753 0 0 0 518 6734 148 27 (2.3) yes 725 0 90 0 225 4140 45 28 (2.4) yes792 0 232 0 754 4814 0 Mean 756.7 0.0 100.5 0.0 424.3 4852.0 48.3 STD33.7 0.0 96.5 0.0 263.0 1333.1 69.8 Group 8 29 (3.1) yes 784 0 153 0 5614233 153 30 (3.2) yes 512 0 152 0 304 6992 152 31 (3.3) yes 701 0 0 0238 3094 68 32 (3.4) yes 631 0 305 0 305 5368 122 Mean 657.0 0.0 152.50.0 352.0 4921.8 123.8 STD 115.1 0.0 124.5 0.0 142.8 1663.3 39.9 Group 933 (4.1) NA NA NA NA NA NA NA NA 34 (4.2) yes 724 0 125 0 540 3780 45 35(4.3) yes 758 0 117 0 429 3315 39 36 (4.4) yes 808 0 47 0 329 4089 235Mean 763.3 0.0 96.3 0.0 432.7 3728.0 106.3 STD 42.3 0.0 42.9 0.0 105.5389.6 111.5 mg/dl mg/dl g/dl g/dl g/dl mg/dl BUN Creatinine T. proteinAlbumin Globulin T. Bilirubin Normal Animal # 13.9-28.3 0.3-1.0 4.0-8.62.5-4.8 1.5-3.8 0.10-0.90 Group 1  1 (0) NA NA NA NA NA NA  2 (0) 28 0.54.9 3.7 1.2 0.3  3 (0) 25 0.5 4.9 3.8 1.1 0.2  4 (0) 27 0.5 4.7 3.7 1  0.2 Mean 26.7 0.5 4.8 3.7 1.1 0.2 STD 1.5 0.0 0.1 0.1 0.1 0.1 Group 2  5(1.5) 34 0.5 4.6 3.6 1   0.2  6 (1.6) 31 0.4 4.6 3.3 1.3 0.2  7 (1.7) 340.6 4.9 4   0.9 0.3  8 (1.8) NA NA NA NA NA NA Mean 33.0 0.5 4.7 3.6 1.10.2 STD 1.7 0.1 0.2 0.4 0.2 0.1 Group 3  9 (2.5) NA NA NA NA NA NA 10(2.6) 33 0.5 4.6 3.6 1   0.3 11 (2.7) NA NA NA NA NA NA 12 (2.8) 31 0.54.8 3.7 1.1 0.2 Mean 32.0 0.5 4.7 3.7 1.1 0.3 STD 1.4 0.0 0.1 0.1 0.10.1 Group 4 13 (3.5) 32 0.7 4.6 3.4 1.2 0.2 14 (3.6) 34 0.4 4.8 3.8 1  0.2 15 (3.7) 30 0.4 4.7 3.4 1.3 0.2 16 (3.8) 24 0.3 5.1 3.8 1.3 0.2 Mean30.0 0.5 4.8 3.6 1.2 0.2 STD 4.3 0.2 0.2 0.2 0.1 0.0 Group 5 17 (4.5) 220.4 4.6 3.3 1.3 0.2 18 (4.6) 31 0.5 4.9 3.7 1.2 0.2 19 (4.7) 23 0.6 4.83.6 1.2 0.2 20 (4.8) 28 0.5 4.5 3.4 1.1 0.2 Mean 26.0 0.5 4.7 3.5 1.20.2 STD 4.2 0.1 0.2 0.2 0.1 0.0 Group 6 21 (1.1) 28 0.3 5.1 3.4 1.7 0.222 (1.2) 36 0.3 5.1 3.8 1.3 0.2 23 (1.3) 32 0.4 4.9 3.5 1.4 0.1 24 (1.4)NA NA NA NA NA NA Mean 32.0 0.3 5.0 3.6 1.5 0.2 STD 4.0 0.1 0.1 0.2 0.20.1 Group 7 25 (2.1) 32 0.2 5   3.4 1.6 0.2 26 (2.2) 24 0.3 4.2 2.8 1.40.1 27 (2.3) 28 0.3 4.8 3.2 1.6 0.2 28 (2.4) 27 0.3 5   3.4 1.6 0.1 Mean27.8 0.3 4.8 3.2 1.6 0.2 STD 3.3 0.0 0.4 0.3 0.1 0.1 Group 8 29 (3.1) 320.3 4.9 3.3 1.6 0.2 30 (3.2) NA NA NA NA NA NA 31 (3.3) 18 0.3 4.8 3.11.7 0.2 32 (3.4) 26 0.2 4.2 2.9 1.3 0   Mean 25.3 0.3 4.6 3.1 1.5 0.1STD 7.0 0.1 0.4 0.2 0.2 0.1 Group 9 33 (4.1) 25 0.2 4.1 2.7 1.4 0.3 34(4.2) NA NA NA NA NA NA 35 (4.3) 23 0.2 4.7 3.1 1.6 0.2 36 (4.4) 29 0.34.7 3.2 1.5 0.3 Mean 25.7 0.2 4.5 3.0 1.5 0.3 STD 3.1 0.1 0.3 0.3 0.10.1 Abbreviations: WBC: white blood cells; RBC: red blood cells; Hg.:hemoglobin; HCT: hematocrit; MCV: Mean corpuscular volume; MCH: meancorpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration;Plt.: platelets; Abs.: Absolute; Baso: basophils; Eos: eosinophils; Abs.Bands: immature neutrophils; Polys: polymorphonuclear cells; Lymph:lymphocytes; Mono: monocytes; BUN: blood urea nitrogen.

Example 14 Elicitation of Human WT1-Specific T-Cell Responses by WholeGene In Vitro Priming

This example demonstrates that WT1 specific T-cell responses can begenerated from the blood of normal individuals.

Dendritic cells (DC) were differentiated from monocyte cultures derivedfrom PBMC of normal donors by growth for 4-10 days in RPMI mediumcontaining 10% human serum, 50 ng/ml GMCSF and 30 ng/ml IL-4. Followingculture, DC were infected 16 hours with recombinant WT1-expressingvaccinia virus at an M.O.I. of 5, or for 3 days with recombinantWT1-expressing adenovirus at an M.O.I. of 10 (FIGS. 21 and 22). Vacciniavirus was inactivated by U.V. irradiation. CD8+ T-cells were isolated bypositive selection using magnetic beads, and priming cultures wereinitiated in 96-well plates. Cultures were restimulated every 7-10 daysusing autologous dendritic cells adeno or vaccinia infected to expressWT1. Following 3-6 stimulation cycles, CD8+ lines could be identifiedthat specifically produced interferon-gamma when stimulated withautologous-WT1-expressing dendritic cells or fibroblasts. TheWT1-specific activity of these lines could be maintained followingadditional stimulation cycles. These lines were demonstrated tospecifically recognize adeno or vaccinia WT1 infected autologousdendritic cells but not adeno or vaccinia EGFP-infected autologousdendritic cells by Elispot assays (FIG. 23).

Example 15 Formulation of RA12-WT1 for Injection: Use of Excipients toStabilize Lyophilized Product

This example describes the formulation that allows the completesolubilization of lyophilized Ra12-WT1.

The following formulation allowed for the recombinant protein Ra12-WT1to be dissolved into an aqueous medium after being lyophylized todryness:

Recombinant Ra12-WT1 concentration: 0.5-1.0 mg/ml; Buffer: 10-20 mMEthanolamine, pH 10.0; 1.0-5.0 mM Cysteine; 0.05% TWEEN®-80(Polysorbate-80); Sugar: 10% Trehalose (T5251, Sigma, Mo.) 10% Maltose(M9171, Sigma, Mo.) 10% Sucrose (S7903, Sigma, Mo.) 10% Fructose (F2543,Sigma, Mo.) 10% Glucose (G7528, Sigma, Mo.).

The lyophilized protein with the sugar excipient was found to dissolvesignificantly more than without the sugar excipient. Analysis bycoomassie stained SDS-PAGE showed no signs of remaining solids in thedissolved material.

Example 16 Formulation of a WT1 Protein Vaccine

This example describes the induction of WT1-specific immune responsesfollowing immunization with WT1 protein and 2 different adjuvantformulations.

According to this example, WT1 protein in combination with MPL®-SEinduces a strong Ab and Interferon-7 (IFN-7) response to WT1. Describedin detail below are the methods used to induce WT1 specific immuneresponses following WT1 protein immunization using MPL®-SE or ENHANZYN®as adjuvant in C57/B6 mice. C57BL/6 mice were immunized with 20 μgrRa2-WT1 combined with either MPL®-SE or ENHANZYN® adjuvants. One groupof control mice was immunized with rRa12-WT1 without adjuvant and onegroup was immunized with saline alone. Three intramuscular (IM)immunizations were given, three weeks apart. Spleens and sera wereharvested 2 weeks post-final immunization. Sera were analyzed forantibody responses by ELISA on plates coated with Ra12-WT1 fusion, Ra12or WT1 TRX. Similar levels of IgG2a and IgG1 antibody titers wereobserved in mice immunized with Ra12-WT1+MPL®-SE and Ra12-WT1+ENHANZYN®.Mice immunized with rRa12-WT1 without adjuvant showed lower levels ofIgG2a antibodies.

CD4 responses were assessed by measuring Interferon-γ productionfollowing stimulation of splenocytes in vitro with rRa12-WT1, rRa12 orwith WT1 peptides p6, p117 and p287. Both adjuvants improved the CD4responses over mice immunized with rRA12-WT1 alone. Additionally, theresults indicate that rRA12-WT1+MPL®-SE induced a stronger CD4 responsethan did rRA12-WT1+ENHANZYN®. IFN-γOD readings ranged from 1.4-1.6 inthe mice immunized with rRA12-WT1+MPL®-SE as compared to 1-1.2 in themice immunized with rRA12-WT1+ENHANZYN®. Peptide responses were onlyobserved against p117, and then only in mice immunized withrRa12-WT1+MPL®-SE. Strong IFN-γ responses to the positive control, ConA,were observed in all mice. Only responses to ConA were observed in thenegative control mice immunized with saline indicating that theresponses were specific to rRA12-WT1.

Example 17 Construction of a Randomly Mutated WT1 Library

The nucleic acid sequence of human WT1 was randomly mutated using apolymerase chain reaction method in the presence of 8-oxo dGTP and dPTP(journal of Molecular Biology 1996; 255:589-603). The complete unsplicedhuman WT1 gene is disclosed in SEQ ID NO:380 and the correspondingprotein sequence is set forth in SEQ ID NO:404. A splice variant of WT1was used as a template for the PCR reactions and is disclosed in SEQ IDNOs:381 (DNA) and 408 (protein). Conditions were selected so that thefrequency of nucleic acid alterations led to a targeted change in theamino acid sequence, usually 5-30% of the PCR product. The mutated PCRproduct was then amplified in the absence of the nucleotide analoguesusing the four normal dNTPs. This PCR product was subcloned intomammalian expression vectors and viral vectors for immunization. Thislibrary, therefore, contains a mixed population of randomly mutated WT1clones. Several clones were selected and sequenced. The mutated WT1variant DNA sequences are disclosed in SEQ ID NOs:377-379 and thepredicted amino acid sequences of the variants are set forth in SEQ IDNOs:405-407. These altered sequences, and others from the library, canbe used as immunogens to induce stronger T cell responses against WT1protein in cancer cells.

Example 18 Construction of WT1-Lamp Fusions

A tripartite fusion was constructed using the polymerase chain reactionand synthetic oligonucleotides containing the desired junctions of humanlysosomal associated membrane protein-1 (LAMP-1) and a splice variant ofthe human WT1 sequence. The splice variant of WT1 and the LAMP-1sequence used for these fusions are disclosed in SEQ ID NOs:381 and 383.Specifically, the signal peptide of LAMP-1 (base pairs 1-87 of LAMP) wasfused to the 5-prime end of the human WT1 open reading frame (1,290 basepairs in length), then the transmembrane and cytoplasmic domain ofLAMP-1 (base pairs 1161 to 1281 of LAMP) was fused to the 3-prime end ofthe WT1 sequence. The sequence of the resulting WT1-LAMP construct isset forth in SEQ ID NO:382 (DNA) and SEQ ID NO:409 (protein). Theconstruct was designed so that when it is expressed in eukaryotic cells,the signal peptide directs the protein to the endoplasmic reticulum (ER)where the localization signals in the transmembrane and cytoplasmicdomain of LAMP-1 direct transport of the fusion protein to the lysosomallocation where peptides are loaded on to Class II MHC molecules.

Example 19 Construction of WT1-Ubiquitin Fusions for Enhanced MHC ClassI Presentation

The human ubiquitin open reading frame (SEQ ID NO:384) was mutated suchthat the nucleotides encoding the last amino acid encode an alanineinstead of a glycine. This mutated open reading frame was cloned inframe just upstream of the first codon of a splice variant of human WT1(SEQ ID NOs:381 and 408, DNA and protein, respectively). The G->Amutation prevents co-translational cleavage of the ancient protein bythe proteases that normally process poly-ubiquitin during translation.The DNA and predicted amino acid sequence for the resulting constructare set forth in SEQ ID NOs:385 and 410, respectively. The resultingprotein demonstrated decreased cellular cytotoxicity when it wasexpressed in human cells. Whereas it was not possible to generate stablelines expressing native WT1, cell lines expressing the fusion proteinwere readily obtained. The resulting protein is predicted to be targetedto the proteosome by virtue of the added ubiquitin molecule. This shouldresult in more efficient recognition of the protein by WT1 specific CD8+T cells.

Example 20 Construction of an Adenovirus Vector Expressing Human WT1

A splice variant of human WT1 (SEQ ID NO:381) was cloned into an E1 andE3 deleted adenovirus serotype 5 vector. The expression of the WT1 geneis controlled by the CMV promoter mediating high levels of WT1 proteinexpression. Infection of human cells with this reagent leads to a highlevel of expression of the WT1 protein. The antigenic nature of theadenoviral proteins introduced into the host cell during and produced atlow levels subsequent to infection can act to increase immunesurveillance and immune recognition of WT1 as an immunological target.This vector can be also used to generate immune responses against theWT1 protein when innoculated into human subjects. If these subjects arepositive for WT1 expressing tumor cells the immune response could have atherapeutic or curative effect on the course of the disease.

Example 21 Construction of a Vaccinia Virus Vector Expressing Human WT1

A splice variant of the full length human WT1 gene (SEQ ID NO:381) wascloned into the thymidine kinase locus of the Western Reserve strain ofthe vaccinia virus using the pSC11 shuttle vector. The WT1 gene is underthe control of a hybrid vaccinia virus promoter that mediates geneexpression throughout the course of vaccinia virus infection. Thisreagent can be used to express the WT1 protein in human cells in vivo orin vitro. WT1 is a self protein that is overexpressed on some humantumor cells. Thus, immunological responses to WT1 delivered as a proteinare unlikely to lead to Major Histocompatibility Class I (MHC classI)-mediated recognition of WT1. However, expression of the protein inthe intracellular compartment by the vaccinia virus vector will allowhigh level MHC class I presentation and recognition of the WT1 proteinby CD8+ T cells. Expression of the WT1 protein by the vaccinia virusvector will also lead to presentation of WT1 peptides in the context ofMHC class II and thus to recognition by CD4+ T cells.

The uses of this invention include its use as a cancer vaccine.Immunization of human subjects bearing WT1 positive tumors could lead toa therapeutic or curative response. The expression of WT1 within thecell will lead to recognition of the protein by both CD4 and CD8positive T cells.

Example 22 Generation of WT1-Specific CD8+ T-Cell Clones Using WholeGene Priming

Dendritic cells (DC) were differentiated from monocyte cultures derivedfrom PBMC of normal donors by growth for 4-6 days in RPMI mediumcontaining 10% human serum, 50 ng/ml GM-CSF and 30 ng/ml IL-4. Followingculture, DC were infected 16 hours with recombinant WT1-expressingvaccinia virus (described in Example 21) at a multiplicity of infection(MOI) of 5 or for 3 days with recombinat WT1-expressing adenovirus at anMOI of 10. Vaccinia virus was inactivated by U.V. irradiation. CD8+T-cells were isolated by negative depletion using magnetic beads, andpriming cultures were initiated in 96-well plates. Cultures wererestimulated every 7-10 days using autologous dendritic cells infectedwith adeno or vaccinia virus engineered to express WT1. Following 4-5stimulation cycles, CD8+ T-cell lines could be identified thatspecifically produced interferon-gamma when stimulated withautologous-WT1 expressing dendritic cells or fibroblasts. These lineswere cloned and demonstrated to specifically recognize WT1 transducedautologous fibroblasts but not EGFP transduced fibroblasts by Elispotassays.

The Wilms' tumor (WT1) gene participates in leukemogenesis and isoverexpressed in most human leukemias as well as in several solidtumors. Previous studies in humans have demonstrated the presence of WT1specific antibody (Ab) responses in 16/63 (25%) of AML and in 15/81(19%) of CML patients studied. Previous studies in mice have shown thatWT1 peptide based vaccines elicit WT1 specific Ab, Th and CTL responses.The use of peptides as vaccines in humans is limited by their HLArestriction and the tendency to elicit peptide specific responses andonly in a minority of patients tumor specific CTL. The advantages ofwhole gene immunization are that several helper and CTL epitopes can beincluded in a single vaccine, thus not restricting the vaccine tospecific HLA types. The data disclosed herein demonstrate the inductionof WT1 specific immune responses using whole gene in vitro priming andthat WT1 specific CD8+ T-cell clones can be generated. Given thatexistent immunity to WT1 is present in some patients with leukemia andthat murine and human WT1 are 96% identical at the amino acid level andvaccination to WT1 protein, DNA or peptides can elicit WT1 specific Ab,and cellular T-cell responses in mice without toxicity to normal tissuesin mice, these human in vitro priming experiments provide furthervalidation of WT1 as a tumor/leukemia vaccine. Furthermore, the abilityto generate WT1 specific CD8+ T-cell clones may lead to the treatment ofmalignancies associated with WT1 overexpression using geneticallyengineered T-cells.

Example 23 Recombinant Constructs for Clinical Manufacturing of WT1

Five constructs were made as described in detail below, for theproduction of clinical grade WT1.

Design of Ra12/WT-E (SEQ ID NOs:388 (cDNA) and 391 (protein)) and WT-1 E(SEQ ID NOs:386 (cDNA) and 395 (protein)) with No His tag:

The WT-1 E reading frame was PCR amplified with the following primersfor the non-His non fusion construct:

PDM-780 (SEQ ID NO: 396) 5′ gacgaaagcatatgcactccttcatcaaac 3′ Tm {tildeover (6)}° C. PDM-779 (SEQ ID NO: 397)5′ cgcgtgaattcatcactgaatgcctctgaag 3′ Tm 63° C.

The following PCR cycling conditions were used: 1 μl 10×Pfu buffer, 1 μl10 mM dNTPs, 2 μl 1M each oligo, 83 μl sterile water 1.5 μl Pfu DNApolymerase (Stratagene, La Jolla, Calif.), 50 μg DNA (pPDMRa12WT-1 NoHis). The reaction was denatured initially at 96° C. for 2 minutes,followed by 40 cycles of 96° C. for 20 seconds, 62° C. for 15 seconds,and 72° C. for 1 minute and 40 seconds. This was followed by a finalextension of 72° C. for 4 minutes. The PCR product was digested withNdeI and EcoRI and cloned into pPDM His (a modified pET28 vector) thathad been digested with NdeI and EcoRI. The construct was confirmedthrough sequence analysis and then transformed into BLR (DE3) pLys S andHMS 174 (DE3) pLys S cells. This construct—pPDM WT-1 E was then digestedwith NcoI and XbaI and used as the vector backbone for the NcoI and XbaIinsert from pPDM Ra12 WT-1 F (see below). The construct was confirmedthrough sequence analysis and then transformed into BLR (DE3) pLys S andHMS174 (DE3) pLys S cells. Protein expression was confirmed by Coomassiestained SDS-PAGE and N-terminal protein sequence analysis.

Design of Ra12-WT-1-F (a.a. 1-281) with No His tag (SEQ ID NOs:389(cDNA) and 393 (protein)):

The Ra12 WT-1 reading frame was PCR amplified with the followingprimers:

PDM-777 (SEQ ID NO: 398) 5′ cgataagcatatgacggccgcgtccgataac 3′ Tm 66° C.PDM-779 (SEQ ID NO: 399) 5′ cgcgtgaattcatcactgaatgcctctgaag 3′ Tm 63° C.

The following PCR cycling conditions were used: 1 μl 10×Pfu buffer, 1 μl10 mM dNTPs, 2 μl 1M each oligo, 83 μl sterile water 1.5 μl Pfu DNApolymerase (Stratagene, La Jolla, Calif.), 50 μg DNA (pPDMRa12 WT-1 NoHis). The reaction was denatured initially at 96° C. for 2 minutes,followed by 40 cycles of 96° C. for 20 seconds, 58° C. for 15 seconds,and 72° C. for 3 minutes. This was followed by a final extension of 72°C. for 4 minutes. The PCR product was digested with NdeI and cloned intopPDM His that had been digested with NdeI and Eco72I. The sequence wasconfirmed through sequence analysis and then transformed into BLR (DE3)pLys S and HMS174 (DE3) pLysS cells. Protein expression was confirmed byCoomassie stained SDS-PAGE and N-terminal protein sequence analysis.

Design of Ra12-WT-1 with No His tag (SEQ ID NOs:390 (cDNA) and 392(protein)):

The Ra12WT-1 reading frame was PCR amplified with the following primers:

PDM-777 (SEQ ID NO: 400) 5′ cgataagcatatgacggccgcgtccgataac 3′ Tm 66° C.PDM-778 (SEQ ID NO: 401) 5′ gtctgcagcggccgctcaaagcgccagc 3′ Tm {tildeover (7)}° C.

The following PCR cycling conditions were used: 1 μl 10×Pfu buffer, 1 μl10 mM dNTPs, 2 μl 1M each oligo, 83 μl sterile water 1.5 μl 1 Pfu DNApolymerase (Stratagene, La Jolla, Calif.), 50 gμDNA (pPDMRa12 WT-1 NoHis). The reaction was denatured initially at 96° C. for 2 minutes,followed by 40 cycles of 96° C. for 20 seconds, 68° C. for 15 seconds,and 72° C. for 2 minutes and 30 seconds. This was followed by a finalextension of 72° C. for 4 minutes. The PCR product was digested withNotI and NdeI and cloned into pPDM His that had been digested with NdeIand NotI. The sequence was confirmed through sequence analysis and thentransformed into BLR (DE3) pLys S and HMS174 (DE3) pLysS cells. Proteinexpression was confirmed by Coomassie stained SDS-PAGE and N-terminalprotein sequence analysis.

Design of WT-1 C (a.a. 69-430) in E. coli without His tag (SEQ IDNOs:387 (cDNA) and 394 (protein)):

The WT-1 C reading frame was PCR amplified with the following primers:

PDM-780 (SEQ ID NO: 402) 5′ gacgaaagcatatgcactccttcatcaaac 3′ Tm {tildeover (6)}° C. PDM-778 (SEQ ID NO: 403) 5′ gtctgcagcggccgctcaaagcgccagc3′ Tm {tilde over (7)}° C.

The following PCR cycling conditions were used: 1 μl 10×Pfu buffer, 1 μl10 mM dNTPs, 2 μl 1M each oligo, 83 μl sterile water 1.5 μl Pfu DNApolymerase (Stratagene, La Jolla, Calif.), 50 μg DNA (pPDMRa12WT-1 NoHis). The reaction was denatured initially at 96° C. for 2 minutes,followed by 40 cycles of 96° C. for 20 seconds, 62° C. for 15 seconds,and 72° C. for 2 minutes. This was followed by a final extension of 72°C. for 4 minutes. The PCR product was digested with NdeI and cloned intopPDM His that had been digested with NdeI and Eco72I. The sequence wasconfirmed through sequence analysis and then transformed into BLR (DE3)pLys S and HMS174 (DE3) pLys S cells. Protein expression was confirmedby Coomassie stained SDS-PAGE and N-terminal protein sequence analysis.

Example 24 Generation of WT1-Specific CD8⁺ T Cell Clones Using WholeGene Priming and Identification of an HLA-A2-Restricted WT1 Epitope

In this example, Adeno and Vaccinia virus delivery vehicles were used togenerate WT1-specific T cell lines. A T cell clone from the line wasshown to be specific for WT1 and further, the epitope recognized by thisclone was identified.

Dendritic cells (DC) were differentiated from monocyte cultures derivedfrom PBMC of normal donors by growth for 4-6 days in RPMI mediumcontaining 10% human serum, 50 ng/ml GM-CSF and 30 ng/ml IL-4. Followingculture, DC were infected 16 hours with recombinant WT1-expressingvaccinia virus at a multiplicity of infection (MOI) of 5 or for 2-3 dayswith recombinant WT1-expressing adeno virus at an MOI of 3-10. Vacciniavirus was inactivated by U.V. irradiation. CD8+ T-cells were isolated bynegative depletion using antibodies to CD4, CD14, CD16, CD19 and CD56+cells, followed by magnetic beads specific for the Fc portion of theseAbs.

Priming cultures were initiated in 96-well plates. Cultures wererestimulated every 7-14 days using autologous dendritic cells infectedwith adeno or vaccinia virus engineered to express WT1. Following 4-5stimulation cycles, CD8+ T cell lines could be identified thatspecifically produced interferon-γ (IFN-γ) when stimulated withautologous-WT1 expressing dendritic cells or fibroblasts. These lineswere cloned and demonstrated to specifically recognize WT1 transducedautologous fibroblasts but not control transduced fibroblasts by Elispotassays.

To further analyze HLA restriction of these WT1 specific CD8+ T-cellclones, fibroblasts derived from an additional donor (D475), sharingonly the HLA-A2 allele with the donor (D349) from which the T-cell clonewas established, were transduced with WT1. ELISPOT analysis demonstratedrecognition of these D475 target cells by the T-cell clone. To furtherdemonstrate HLA A2 restriction and demonstrate that this epitope isexpressed by tumor cells “naturally” overexpressing WT1 (as part oftheir malignant transformation), the leukemia cell line K562 was tested.K562 was transduced with the HLA A2 molecule, and HLA-A2 negative K562cells were used as controls for nonspecific IFN-γ release. ELISPOTanalysis demonstrated that the T cells recognized the A2 positive K562cell line, but not the A2 negative K562 cells. Further proof ofspecificity and HLA-A2 restriction of the recognition was documented byHLA-A2 antibody blocking experiments.

To further define the WT1 epitope, 4 truncated WT1 retroviral constructswere generated. Donor 475 fibroblasts were then transduced with theseconstructs. ELISPOT assays demonstrated recognition of D475 fibroblaststransduced with the WT1 Tr1 construct (aa2-aa92), thus demonstratingthat the WT1 epitope is localized within the first 91 N-terminal aminoacids of the WT1 protein. To fine map the epitope, 15mer peptides of theWT1 protein, overlapping by 11 amino acids, were synthesized. The WT1specific T-cell clone recognized two overlapping 15mer peptides, peptide9 (QWAPVLDFAPPGASA) (SEQ ID NO: 412) and peptide 10 (VLDFAPPGASAYGSL)(SEQ ID NO: 413). To further characterize the minimal epitoperecognized, shared 9mer and 10 mer peptides of the 15mers (5 total) wereused to analyse the specificity of the clone. The clone specificallyrecognized the 9mer, VLDFAPPGA (SEQ ID NO:241), and the 10 mer,VLDFAPPGAS (SEQ ID NO:411).

Example 25 Cloning and Sequencing of TCR Alpha and Beta Chains Derivedfrom a CD8 T Cell Specific for WT1

T cell receptor (TCR) alpha and beta chains from CD8+ T cell clonesspecific for WT1 are cloned. Sequence analysis is carried to demonstratethe family origin of the alpha and beta chains of the TCR. Additionally,unique diversity and joining segments (contributing to the specificityof the response) are identified.

Total mRNA from 2×10⁶ cells from a WT1 specific CD8+ T cell clone isisolated using TRIZOL® reagent (a mono-phasic solution of phenol andguanidine isothiocyanate) and cDNA is synthesized using READY-TO-GO®kits (Pharmacia). To determine Vα and Vβ sequences in a clone, a panelof Vα and Vβ subtype specific primers are synthesized (based on primersequences generated by Clontech, Palo Alto, Calif.) and used in RT-PCRreactions with cDNA generated from each clone. The RT-PCR reactionsdemonstrate which Vβ and Vα sequence is expressed by each clone.

To clone the full-length TCR alpha and beta chains from a clone, primersare designed that span the initiator and terminator-coding TCRnucleotides. Standard 35 cycle RT-PCR reactions are established usingcDNA synthesized from the CTL clone and the above primers using theproofreading thermostable polymerase PWO (Roche, Basel, Switzerland).The resultant specific bands (˜850 bp for alpha and ˜950 for beta) areligated into the PCR blunt vector (Invitrogen, Carlsbad, Calif.) andtransformed into E. coli. E. coli transformed with plasmids containingfull-length alpha and beta chains are identified, and large scalepreparations of the corresponding plasmids are generated. Plasmidscontaining full-length TCR alpha and beta chains are then sequencedusing standard methods. The diversity-joining (DJ) region thatcontributes to the specificity of the TCR is thus determined.

Example 26 WT1 Specific CD8+ T-Cell Clone Lyses WT1-Expressing LeukemicBlasts

The CD8+ T cell clone initially disclosed in Example 24 that recognizespeptide sequence VLDFAPPGA (human WT1 residues 37-45; SEQ ID NO:241) wasfurther tested for the ability to kill (lyse) WT1 expressing leukemiatarget cells in an HLA A2 restricted fashion. K562 target cellstransduced with the HLA A2 molecule, GFP, A2 Kb, or untransduced, wereused in a standard 4.5 hour ⁵¹Chromium release assay with effector totarget cell (E:T) ratios of 25:1 and 5:1. At an E:T ratio of 25:1, theCD8+ T-cell clone lysed the K562/A2 and K562/A2 Kb cells (40% and 49%specific lysis, respectively) while the control GFP transduced and theK562 cells were not lysed. At an E:T of 5:1, specific lysis of theK562/A2 and K562/A2 Kb cells was 21% and 24%, respectively. Thus, thisCD8+ T cell clone recognizes and lyses leukemic cells expressing WT1 inan HLA-A2-restricted fashion. The ability to generate WT1 specific CD8+T-cell clones has utility in the treatment of malignancies associatedwith WT1 overexpression using genetically engineered T-cells.

Example 27 Construction of HLA-A2-Peptide-MHC Tetrameric Complexes

This example describes the cloning and expression of soluble HLA-A2 ininsect cells, and the purification and assembly of HLA-A2 intofluorescent, multivalent peptide-MHC tetramer complexes for thedetection and isolation of antigen-specific CD8 T cells.

This system is similar to that developed and described by Altman, et al.(Altman, J., et al., Science, 1996 274(5284):94-6) in that solubleHLA-A2 was singly biotinylated at a birA recognition sequence and wassubsequently assembled into multimers on a phycoerythrin-conjugatedstreptavidin scaffolding. The materials described herein differ in thatthe HLA-A2 was expressed in a glycosylated, soluble form from insectcells and the heterodimer was purified using an anti-human class I MHCantibody affinity column.

The HLA-A2 heavy chain gene, appended with the birA biotinylationsequence, and the human beta-2-microglobulin gene were cloned into thebaculovirus expression vector pFASTBAC-dual. Upon infection of insectcells the genes were concomitantly transcribed from divergent promotersand fully assembled, glycosylated soluble HLA-A2 heterodimer wassecreted into the growth medium. The infected insect cells were culturedin cell factories for 4 days at 21° C. before the supernatants wereharvested. HLA-A2 production was monitored by a capture ELISA employingthe W6/32 and biotinylated B9.12.1 antibodies. HLA-A2 was purified fromthe culture supernatant to >90% purity in one step by affinitychromatography using 2 anti-human class I MHC monoclonal antibodieslinked to Sepharose beads. The antibodies used were PA2.1 and W6/32.Purified HLA-A2 was singly biotinylated on the birA recognition sequenceon the C-terminus of the heavy chain using the commercially availablebirA enzyme. The efficiency of biotinylation was evaluated essentiallyas described (Crawford et al (1998) Immunity June; 8(6):675-82.), andthe material was further purified by size exclusion chromatography(SEC). Phycoerythrin-conjugated streptavidin was saturated withbio-HLA-A2 and the multivalent staining reagent was purified from freeHLA-A2 by SEC. HLA-A2 tetramer was incubated for 48 hours at roomtemperature with a 10-fold molar excess of Her-2/neu E75 peptide orInfluenza matrix MI peptide before the specific T cell clones werestained at 4° C. for 30 minutes in the presence of peptide loadedtetramer and anti-CD8 antibody. Results indicated that the tetramersincubated in the presence of molar excess of the M158-66 M1 influenzapeptide specifically stained an influenza-specific T cell clone and thetetramers incubated with an excess of the Her-2/neu E75 peptidespecifically stained the Her-2/new specific T cell clone.

Example 28 Detection of WT1 Specific T-Cells Using WT1 MHC-PeptideTetramers

HLA-A2 tetramers described in Example 27 were incubated with a molarexcess of the WT1 p37-45 peptide (VLDFAPPGA) (human WT1 residues 37-45;SEQ ID NO:241) previously shown in Example 24 to be restricted byHLA-A2. This tetramer was used to stain the WT1-specific CD8+ T cellclone described in Example 24. This clone was shown to specificallyrecognize the p37-45 epitope. When the tetramers were incubated with anexcess of p37-45 peptide, they specifically stained the CD8+ T cellclone while those tetramers incubated with an excess of irrelevantHLA-A2 peptides (Her2/neu, WT1 p38-46, WT1 p39-47), the tetramers didnot stain the CD8+ T cell clone. Thus, the WT1 p37-45-specific CD8+ Tcell clone is specifically recognized by the HLA-A2-p37-45 peptide MHCtetramer.

A WT1-specific T cell line generated as described in Example 24 was thenstained with the HLA-A2-p37-45, irrelevant Her2/neu or WT1 p37-46tetramers. The HLA-A2-p37-45 tetramers stained 1% of the totalpopulation of this WT1-specific T cell line and 7% of the gated CD8+population while the control HLA-A2-p37-46 tetramer stained at the samebackground levels as the control HLA-A2-Her2/neu tetramers.

These results indicate that MHC-peptide tetramers are a highly sensitiveand specific tool for detecting WT1 specific immune responses. Thepeptide-MHC tetramers can be used for early detection of WT1 associatedmalignancies, monitoring WT1-specific responses, and for monitoringminimal residual disease. Detection of WT1 specific T-cells by tetramerstaining is also a useful tool to identify groups within a patientpopulation suffering from a WT1 associated disease at a higher risk forrelapse or disease progression.

Example 29 Generation of a WT1-Specific CD8⁺ T Cell Line from anHLA-A24-Positive Donor Using Whole Gene Priming

In this example, Adeno and Vaccinia virus delivery vehicles were used togenerate WT1-specific T cell lines from an HLA-A24 positive donor. ThisT cell line was shown to be MHC class I restricted. These experimentsfurther confirm the immunogenicity of the WT1 protein and support itsuse as a target for vaccine and/or other immunotherapeutic approaches.

Dendritic cells (DC) were differentiated from monocyte cultures derivedfrom PBMC of a normal HLA-A24-positive donor by growth for 4-6 days inRPMI medium containing 10% human serum, 50 ng/ml GM-CSF and 30 ng/mlIL-4. Following culture, DC were infected 16 hours with recombinantWT1-expressing vaccinia virus at a multiplicity of infection (MOI) of 5or for 2-3 days with recombinant WT1-expressing adeno virus at an MOI of3-10. Vaccinia virus was inactivated by U.V. irradiation. CD8+ T-cellswere isolated by negative depletion using antibodies to CD4, CD14, CD16,CD19 and CD56+ cells, followed by magnetic beads specific for the Fcportion of these Abs.

Priming cultures were initiated in 96-well plates. Cultures wererestimulated every 7-14 days using autologous dendritic cells infectedwith adeno or vaccinia virus engineered to express WT1. Following 4-5stimulation cycles, CD8+ T cell lines could be identified thatspecifically produced interferon-γ (IFN-γ) when stimulated withautologous-WT1 expressing dendritic cells or fibroblasts. These lineswere cloned and shown by Elispot assays to specifically recognize WT1transduced autologous fibroblasts but not control transduced fibroblastsin an MHC class I-restricted manner.

These experiments show that the WT1 protein can be used to generate a Tcell response and thus, further confirm the immunogenicity of the WT1antigen and support its use as a target for vaccine and otherimmunotherapeutic approaches.

Example 30 Identification of HLA-A2 High Affinity WT1 Epitopes

This experiment describes the in silico identification of WT1 epitopespredicted to bind to HLA-A2 with higher affinity than naturallyprocessed epitopes. The epitopes identified herein have utility invaccine and/or immunotherapeutic strategies for the treatment of cancersassociated with WT1 expression.

Peptide analogs of the naturally processed HLA A2 restricted WT1 epitopep37-45 (VLDFAPPGA; human WT1 residues 37-45; SEQ ID NO:241; previouslyshown in Example 24 to be restricted by HLA-A2) with motifs for bindingto HLA-A2.1 with higher affinity than the naturally processed peptidewere constructed as described in further detail below.

A peptide motif searching program based on algorithms developed byRammensee, et al (Hans-Georg Rammensee, Jutta Bachmann, Niels NikolausEmmerich, Oskar Alexander Bachor, Stefan Stevanovic: SYFPEITHI: databasefor MHC ligands and peptide motifs. Immunogenetics (1999) 50: 213-219)and by Parker, et al (Parker, K. C., M. A. Bednarek, and J. E. Coligan.1994. Scheme for ranking potential HLA-A2 binding peptides based onindependent binding of individual peptide side-chains. J. Immunol.152:163.) was used to identify analogs of the WT1 p37-45 peptide epitopethat are predicted to bind to HLA-A2 with higher affinity than thenatural p37-45 peptide. The peptides shown in Table LII have predictedpeptide binding scores equal to or greater than the naturally processedp37-45 peptide. The binding score is derived from a predicted half-timeof dissociation to the HLA-A2 class I molecule. The analysis is based oncoefficient tables deduced from the published literature by Dr. KennethParker kparker@atlas.niaid.nih.gov, NIAID, NIH.

TABLE LII p37-45 Peptide Analogs Position Theoretical Binding SEQ IDModified Sequence Score NO: Wild Type VLDFAPPGA 3.378 241 P1 ILDFAPPGA3.378 414 P1 LLDFAPPGA 3.378 415 P1 FLDFAPPGA 9.141 416 P1 KLDFAPPGA6.955 417 P1 MLDFAPPGA 3.378 418 P1 YLDFAPPGA 9.141 419 P2 VMDFAPPGA2.44 420 P4 VLDEAPPGA 13.85 421 P4 VLDKAPPGA 3.378 422 P6 VLDFAVPGA 7.77423 P8 VLDFAPPKA 3.378 424 P9 VLDFAPPGV 47.3 425 P9 VLDFAPPGL 14.53 426P1 and P4 FLDEAPPGA 37.48 427 P1 and P4 KLDEAPPGA 28.52 428 P1 and P4YLDEAPPGA 37.48 429 P1 and P4 FLDKAPPGA 9.141 430 P1 and P4 KLDKAPPGA6.955 431 P1 and P4 YLDKAPPGA 9.141 432 P1 and P9 FLDFAPPGV 128 433 P1and P9 KLDFAPPGV 97.37 434 P1 and P9 YLDFAPPGV 128 435 P1 and P9FLDFAPPGL 39.31 436 P1 and P9 KLDFAPPGL 29.91 437 P1 and P9 YLDFAPPGL39.31 438 P1, P4 and P9 FLDEAPPGV 524.7 439 P1, P4 and P9 KLDEAPPGV399.2 440 P1, P4 and P9 YLDEAPPGV 524.7 441 P1, P4 and P9 FLDEAPPGL161.2 442 P1, P4 and P9 KLDEAPPGL 122.6 443 P1, P4 and P9 YLDEAPPGL161.2 444

In a separate analysis, computer modeling was used to identify peptideepitope analogs of the p37-45 WT1 epitope. The coordinates of the HLA-A2native structure were downloaded from the Brookhaven protein database(pdb I.D.: 3HLA) (L. L. Walsh, “Annotated PDB File Listing”, ProteinScience 1:5, Diskette Appendix (1992). This file was used as a templatefor manipulations with the SwissModel (Peitsch MC (1996) ProMod andSwiss-Model: Internet-based tools for automated comparative proteinmodeling. Biochem. Soc. Trans. 24:274-279.) program available throughthe Expasy web site (Appel R. D., Bairoch A., Hochstrasser D. F. A newgeneration of information retrieval tools for biologists: the example ofthe ExPASy WWW server. Trends Biochem. Sci. 19:258-260(1994). Thepeptide bound to the protein was mutated manually to yield the bound WTp37-45 peptide. The new structure was submitted for three rounds ofenergy minimization with the GROMOS96 implementation of theSwiss-PdbViewer; two energy minimizations were performed on the wholestructure, followed by one round with unfavorable residues selected. Afinal evaluation showed an overall favorable energy state for the model.Ramachandran plotting indicated that only one non-glycinyl residue isfar in disallowed regions. Peptides identified using the modeling methoddescribed herein are set forth in Table LIII below.

TABLE LIII p37-45 Peptide Analogs Identified by Computer ModelingPosition SEQ ID Modified Sequence NO: Wild Type VLDFAPPGA 241 P6VLDFAGPGA 445 P6 VLDFATPGA 446 P6 and P9 VLDFATPGV 447 P6 and P9VLDFATPGL 448 P6 and P9 VLDFATPGS 449 P6 and P9 VLDFATPGA 450

Several peptides identified using the two methods described above werethen tested for the ability to be recognized by the p37-45 specific CTLclone (see Example 24). ELISPOT analysis showed that peptides p37-1 (SEQID NO:426) and model-1 (SEQ ID NO:445) were recognized by the p37-45 CTLclone. These results suggest that these 2 peptide analogs are predictedto bind to HLA-A2 with higher affinity than the naturally processedepitope and still be recognized by a native T cell receptor.

Thus, this experiment describes the in silico identification of WT1epitopes predicted to bind to HLA-A2 with higher affinity than naturallyprocessed epitopes. Two of the epitopes identified were tested and shownto be recognized by a CTL clone generated with the native WT1 p37-45epitope. The epitopes identified herein have utility in vaccine and/orimmunotherapeutic strategies for the treatment of cancers associatedwith WT1 expression.

Example 31 The In Vivo Immunogenicity of the WT1 Antigen

This example describes three in vivo immunogenicity studies to evaluatevaccination strategies with WT1 in mice. The three strategiescomprised: 1) a naked DNA vaccine prime and boost; 2) an attenuatedadenovirus prime followed by an attenuated alphavirus boost; or 3) anaked DNA prime followed by an adenovirus boost. The full-length cDNA ofthe splice variant of WT1 used in these studies is set forth in SEQ IDNO:381. The results described herein provide support for the use of WT1DNA/DNA, DNA/adenovirus or adenovirus/alphavirus prime/boost regimens asvaccine strategies for treating cancers associated with WT1 expression.

In the first study, C57/BI6 mice were immunized 3 times at 2 weekintervals with 100 μg of naked DNA encoding for WT1. Mice weresacrificed 2-3 weeks after the final immunization and CTL were evaluatedby standard Chromium release assay. This first study showed that WT1 DNAimmunization elicits WT1-specific cytotoxic T cell responses in thesemice with a 25:1 E:T ratio showing 40% lysis.

In the second study, HLA-A2/Kb transgenic mice were immunized once with5×10⁸ PFU of attenuated adenovirus encoding WT1 (as described in Example20) followed 4 weeks later by one boost with 5×10⁶ PFU of alphavirus(AlphaVax) encoding WT1. Mice were sacrificed 2-3 weeks after the finalimmunization and CTL were evaluated by standard Chromium release assay.The results showed that WT1-specific CTL in HLA-A2/Kb transgenic micespecifically lysed dendritic cells (DC) transduced with WT1-expressingviral construct as well as DC pulsed with WT1 peptides. Thus, thisimmunization strategy also effectively elicits WT1-specific CTL in vivo.

In the third study, C57/BI6 and HLA-A2/Kb transgenic mice were immunizedtwice with 100 μg of naked WT1 DNA 2 weeks apart followed 3 weeks laterby a boost with 7×10⁸ PFU adenovirus encoding WT1. Mice were sacrificed2-3 weeks after the final immunization and CTL were evaluated by IFN-γELISPOT assay. The results showed that the WT1 DNA and adenovirusprime-boost generates a WT1-specific CD8 T cell response in HLA-A2/Kbtransgenic mice. About 42% of CD8 positive cells stained positive forIFN-γ following a 7 day stimulation with DCs transduced with WT-1. Theresults from the C57/BL6 mice showed that this immunization strategygenerates CD8 responses detectable in fresh splenocytes. Splenocyteswere stimulated for 6 hours with pools of 10 15-mer peptides overlappingby 11 amino acids that span the entire WT1 protein. Only cellsstimulated with the p121-171 showed IFN-γ staining. About 1.1% of thoseCD8 T cells stimulated with the p121-171 peptide pool stained positivefor IFN-γ. This peptide contains the p117-139 peptide (SEQ ID NO:2)shown in Example 3 to elicit CTL, T helper cell and antibody responsesin mice.

In summary, these results show that the three immunization strategiestested herein generate T cell responses in vivo. Thus, these studiesfurther confirm the immunogenicity of the WT1 protein and providesupport for the use of WT1 DNA/DNA, DNA/adenovirus oradenovirus/alphavirus prime/boost regimens as vaccine strategies fortreating cancers associated with WT1 expression.

Example 32 Reduction in WT1+Tumor Growth in HLA-A2/kb Transgenic MiceImmunized with WT1 Protein

This example describes the reduction of WT1+ tumors in transgenic miceimmunized with a WT1 vaccine. These results further validate WT1 as avaccine target and provide support for the use of WT1 in vaccinestrategies for treating cancers associated with WT1 expression.

The murine dendritic cell (DC) line DC2.4. was stably transduced with aWT1-LAMP construct (see Example 18, cDNA and protein sequences set forthin SEQ ID NO:382 and 409, respectively). Mice were then inoculatedeither subcutaneously (s.c.) or intraperitoneally (i.p.) with 2×10⁶cells. This resulted in tumor growth in 80-100% of the mice. The tumorsestablished in mice in vivo retained their WT1 expression. Thus, thismodel provides a system in which to validate the efficacy of WT1 vaccinestrategies.

Three groups of A2/Kb mice were then immunized 3 times, 2 weeks apart asfollows:

Group 1: saline alone s.c. (control, n=10 mice)

Group 2: MPL®-SE 10 μg alone s.c. (control, n=10 mice)

Group 3: Raa12/WT1 protein 100 μg+10 μg MPL®-SE s.c. (n=9 mice)

Two to three weeks after the last WT1 immunization, mice were inoculatedwith 2×10⁶ A2/Kb DC2.4 tumor cells overexpressing WT1. After tumorchallenge mice were monitored and tumor size measured twice per week upto 4 weeks after tumor challenge.

The results showed that the percentage of mice with tumor growth in thegroup that received the WT1 protein vaccine was reduced from about 100%(saline control) or 90% (MPL®-SE adjuvant control) to 45% (WT1 proteinimmunized group). Further, the average tumor volume was reduced in thisgroup from an average tumor size of 1233 cmm (saline control) or 753 cmm(MPL®-SE adjuvant control) observed in the control group to 226 cmm inthe WT1 protein immunized group. Histopathological analyses showed thattumor margins in vaccinated animals were mixed with host immunologicalreactions including histiocytes, eosinophils, lymphocytes, mast cellsand plasmacytes. Taken together, the results demonstrate that WT1protein immunization protects against or delays the growth ofWT1-positive tumors in the animals immunized with WT1. Thus, theseresults support the use of WT1 protein as a vaccine for malignanciesassociated with WT1 expression.

Example 33 Identification of a Naturally Processed WT1 Cytotoxic T CellEpitope

This example describes the identification of a naturally processedepitope of the WT1 protein recognized by cytotoxic T cells. Thisexperiment further confirms the immunogenicity of the WT1 protein andprovides support for its use as a target for vaccine and/or otherimmunotherapeutic approaches. Additionally, this experiment identifiesepitopes of the WT1 protein that may be used in these applications.

HLA-A2/Kb transgenic mice were immunized twice with 100 ug of naked WT1DNA 2 weeks apart followed 3 weeks later by a boost with 10⁷ PFUadenovirus encoding WT1. Mice were sacrificed 2-3 weeks after the finalimmunization and CTL were evaluated by standard chromium release assay.As observed in previous experiments, immunization with WT1 DNA followedby adenoviral boost elicited a WT1-specific CTL response in HLA-A2transgenic mice. In order to identify which epitopes were recognized bythe T cells, CTL lines were generated and cloned by limiting dilutionusing standard protocols. A positive clone was then tested using astarget cells DC2.4 A2/Kb cells pulsed with peptides corresponding to thetop 20 predicted HLA-A2 restricted CTL epitopes. The results showed thatthe WT1 p10-18 9mer peptide (amino acids: ALLPAVPSL, set forth in SEQ IDNO:451) was recognized by this CTL clone. This epitope was previouslypredicted to be an epitope, as described in Table XLVI, SEQ ID NO:34. Inan additional experiment, CTL responses to the p10 peptide were observedin 4 of 5 WT1 immunized animals tested. Thus, this experimentdemonstrates that the predicted p10-18 WT1 epitope is naturallyprocessed and recognized by CTLs. Moreover, this experiment confirms theimmunogenicity of the WT1 protein and further defines a naturallyprocessed HLA-A2-restricted CTL epitope that can be used in vaccine andimmunotherapeutic strategies for the treatment of malignanciesassociated with WT1 overexpression.

Example 34 WT1 Expression Constructs Using Twin Arginine Translocator(TAT) Signal Peptide

This example describes the construction of WT1-TAT vectors andexpression of WT1-TAT from these vectors. These constructs have utilityin the expression of WT1-TAT molecules for the use in vaccinationstrategies.

WT-1-F (a.a. 2-281 of the WT1 protein; cDNA and amino acid sequence of2-281 of WT1 are set forth in SEQ ID NOs:460 and 461, respectively) andfull-length WT-1 were constructed as pTAT fusions with no His tag asdescribed below. The cDNA sequence of the resulting fusion contructspTAT-WT1F and the pTAT-WT1 full-length are set forth in SEQ ID NOs 452and 453 respectively. The amino acid sequences of the resulting fusionsare set forth in SEQ ID NOs:455 and 454, respectively.

The WT-1-F open reading frame was PCR amplified with the followingprimers:

PDM-439 (SEQ ID NO: 456): 5′ GGCTCCGACGTGCGGGACCTGAAC 3′ Tm 66° C.PDM-779 (SEQ ID NO: 457): 5′ CGCGTGAATTCATCACTGAATGCCTCTGAAG 3′ Tm63° C.

The WT-1 full-length open reading frame was amplified with the followingprimers:

p37 (SEQ ID NO: 458): 5′ GGCTCCGACGTGCGGGACCTG 3′ p23 (SEQ ID NO: 459):5′ GAATTCTCAAAGCGCCAGCTGGAGTTTGGT 3′

The PCR conditions were as follows: 1 μl 10×Pfu buffer, 1 μl 10 mM dNTPs2 μl 1 μM each oligo 83 μl sterile water 1.5 μl Pfu DNA polymerase(Stratagene, La Jolla, Calif.) 50 ng DNA (pPDM FL WT-1). The reactionwas denatured at 96° C. for 2 minutes followed by 40 cycles of 96° C.for 20 seconds, 64° C. for 15 seconds, and 72° C. for 2 minutes, 30seconds and a single, final extension of 4 minutes at 72° C.

The PCR products were digested with EcoRI and cloned into pTAT (amodified pET28 vector with a Twin Arginine Translocation (TAT) signalpeptide from the TorA signal peptide in E. coli on the N-terminus; seeJ. Mol. Microbiol. (2000) 2(2): 179-189; Journal of Bacteriology,January 2001 p604-610 Vol 183, No 2; Journal of Biochemistry Vol 276,Mar. 16, 2001 pp 8159-8164) at the Eco72I and EcoRI sites. The sequenceswere confirmed through sequence analysis and then transformed into BLR(DE3) pLys S and HMS174 (DE3) pLysS cells. Expression of the WT1-TATproteins was confirmed by Western analysis.

Example 35 The N-Terminus of WT is the Dominant In Vivo ImmunogenicPortion of the Protein

In this Example, mice were immunized with different protein constructsof WT-1, (F truncation (2-281) and full length (2-430) as described inExample 34)) formulated with MPL®-SE adjuvant. Improved CD4 responseswere elicited by the truncated constructs relative to the full lengthprotein. Thus, this example demonstrates that the N-terminal portion ofthe WT1 protein spanning from amino acid 2 to 281 is the dominantimmunogenic portion of the WT1 protein in vivo.

Groups of four C57BL/6 mice were immunized subcutaneously with 20 μgWT-1 proteins: WT-1-F or WT-1 full length (FL), with Ra12, HIS or TATfusions. Immunizations were performed at weeks 0, 3 and 9, and spleenswere harvested at week 11. Splenocytes were then stimulated in vitro for6 hours with medium alone, with a 15-mer peptide “p32” (ARMFPNAPYLPSCLE,amino acids 125-139 of WT-1; found within the p117-139 peptide set forthin SEQ ID NO:2), with the DC2.4-WT-1/LAMP cell line, or with rRa12. CD4cells were then stained for intracellular interferon-gamma andquantified by FACS analysis. A portion of these splenocytes were thenstimulated in vitro for 8 days, after which CD4+ IFN+ cells wereenumerated. After the 6 hour stimulation with p32, 0.33% of CD4-positivecells were positive for intracellular IFN-gamma staining in miceimmunized with the truncated N-terminal construct rWT1-F-TAT. Bycontrast, only 0.10% of CD4-positive cells stained positive forintracellular IFN-gamma in mice immunized with rWT1-FL-TAT. After the 8day stimulation, mice immunized with the rWT1-F-TAT construct showedIFN-gamma staining in 10.72% of the CD4+ cells. By contrast, 0.24% ofCD4-positive cells from mice immunized with the full-length WT1-TATconstruct stained positive for intracellular IFN-gamma. These dataindicate that improved CD4 responses were elicited by the truncatedrWT1-TAT construct relative to the full-length rWT1-TAT construct.

In a second assay splenocytes were stimulated in vitro with the 23-merpeptide, p117-139 (SEQ ID NO:2; PSQASSGQARMFPNAPYLPSCLE, containing aknown CD4 epitope and encompassing “p32”), for 3 days, after whichsupernatants were assayed for secreted IFN-gamma by ELISA. There was nodetectable IFN-gamma secretion from splenocytes from mice immunized withthe full-length WT1 constructs. By contrast, an average of 2477 pg/mlIFN-gamma was detected from splenocytes from mice immunized with rWT1-Fwithout a HIS tag. An average of 4658 pg/ml IFN-gamma was detected fromsplenocytes from mice immunized with rWT1-F-TAT. These data furthersupport the observation that improved CD4 responses were elicited by thetruncated N-terminal WT1 constructs relative to the full length protein.

The WT1 protein is a transcription factor which is composed of twofunctional domains: a proline-glutamine rich domain at the N-terminus,and a zinc finger domain composed of four zinc fingers at the C-terminuswith homology to the EGR1/Sp1 family of transcription factors. WT1 is aself-protein. The C-terminus is homologous to other self-proteins and isthus less immunogenic, i.e. the subject of a greater degree ofimmunological tolerance. Of note, the 4 zinc-finger domains within theC-terminus have homology to EGR family members. The results described inthis example indicate that tolerance will vary between differentportions of a protein, possibly depending on sequence homologies andfunctional domains.

In summary, the data described in this example support the notion thatthe most efficient WT1 vaccine will comprise the WT1 N-terminus, eitheras a recombinant protein or gene-based construct.

Example 36 Baculovirus Expression Constructs for Expression of theN-Terminal Fragment of WT1 (WT-1-F: Amino Acids 1-281) and Large ScaleProduction of Protein Using Insect Cells

The cDNA for the N-terminal fragment of WT-1, together with a Kozakconsensus sequence, were obtained by PCR using the WT1-F plasmid as atemplate (WT-1-F: amino acids 2-281 of the WT1 protein cloned downstreamof a start methionine; cDNA and amino acid sequence of 2-281 of WT1 areset forth in SEQ ID NOs:460 and 461, respectively. The pTAT fusionconstruct used as template in the experiments described herein isdescribed in Example 34. The cDNA of this construct is set forth in SEQID NO:452). The following primers were used for amplification:

WT1F1 (SEQ ID NO: 466): 5′ CGGCTCTAGAGCCGCCACCATGGGCTCCGACGTGCG WT1RV4(SEQ ID NO: 467): 5′ CGGCTCTAGACTACTGAATGCCTCTGAAGACACCGTG

The cDNA for the same ORF plus a C-terminal 10 residue His Tag wasobtained by PCR similarly as above except using WT1 RV3 (SEQ ID NO:468)as reverse primer (5′CGGCTCTAGACTAATGGTGATGGTGATGATGATGGTGATGATGCTGAATGCC TCTGAAGACACCGTG).

The purified PCR products were cloned into the Xba I site of the donorplasmid, pFastBac1. The recombinant donor plasmid pFBWT1-F (cDNA andamino acid sequences set forth in SEQ ID NOs:463 and 465, respectively)and pFBWT1-FH (with the 10×His Tag; cDNA and amino acid sequence setforth in SEQ ID NOs:462 and 464, respectively) were transformed into E.coli strain DH10Bac (Invitrogen, Carlsbad, Calif.) to make recombinantbacmids in E. coli through site-specific transposition. The recombinantbacmids were confirmed by PCR analysis and then transfected into Sf-9insect cells to make recombinant baculoviruses BVWT1-F and BVWT1-FH. Therecombinant viruses were amplified to high titer viral stock in Sf-9cells.

The HIGH FIVE insect cell line was used to optimize conditions for theprotein expression and for the large-scale production of the recombinantproteins. To optimize the conditions for protein expression, High 5insect cell monolayers were infected with the recombinant baculovirusesBVWT1-F and BVWT1-FH at different multiplicities of infection (MOI) andharvested the transduced cells at different periods of time. Theidentities of the proteins were confirmed by Western blot analysis witha rabbit anti-WT1 polyclonal antibody [#942-32 (799L)]. Both WT1-F andWT1-FH recombinant proteins were well expressed at either 48 hours or 54hours post-infection when HIGH FIVE cells were infected by therecombinant viruses at MOI 1.0 or 2.0.

The amplification of the recombinant baculoviruses and the expression ofthe recombinant WT1-F and WT1-FH proteins was then further optimized.Both BVWT1-F and BVWT1-FH were amplified in the Sf9 insect cell line inESF921 medium containing 2% fatal calf serum and 0.5×PSF. Theamplification was carried out for 4 days at MOI 0.05 at 28° C. The HIGHFIVE insect cell line was used to optimize conditions for the proteinexpression. High 5 insect cells were infected by the recombinantbaculoviruses at MOIs of 0.5, 1 or 2 for 30, 48, 54, or 72 hours beforeharvesting. The identity of the recombinant protein was confirmed byWestern blot analysis with a specific rabbit polyclonal antibody againstWT1 (Antibody #942-32), and by capillary LC-ESI-tandem massspectrometry. These experiments indicate that optimal protein expressionfor large scale production of WT1-F and WT1-FH occurs at 43 hourspost-infection when HIGH FIVE cells were infected by the recombinantviruses at an MOI 0.5-1.0.

In summary, the above WT1 baculovirus can be used for large-scaleprotein production of the N-terminal portion of WT1 for use in a varietyof vaccine strategies for the treatment of malignancies associated withWT1 expression.

Example 37 Induction of In Vivo CD4 and CD8 T Cell Responses in MiceUsing Recombinant Tricom Vaccinia and Fowl Pox Vectors

This example describes in vivo immunogenicity studies to evaluatevaccination strategies with WT1 in mice. The purpose of theseexperiments was to test the ability to rV-WT1/TRICOM® and rF-WT1/TRICOM®to induce immunity, in particular T cell immunity, to WT1. The resultsdescribed herein provide support for the use of TRICOM® vaccinia andfowlpox vectors expressing WT1 and containing a triad of costimulatorymolecules (B7-1, ICAM-1 and LFA-3) in vaccine strategies for treatingcancers associated with WT1 expression.

In the first study, C57BI/6 mice (12 mice per group) were immunized twoor three times with 14 days between the primary, secondary and tertiaryimmunizations as shown below in Table 1. Mice were harvested at 21 daysfollowing the secondary and tertiary immunizations. CD8 and CD4 T cellresponses were assayed by IFN-γ intracellular cytokine staining ofWT1-peptide activated spleen cells as described in further detail below.CD4 T cell responses were additionally assayed by IFN-γ release fromrWT1 protein stimulated spleen cells. Serum IgG₁ and IgG_(2b) antibodyresponses were assayed by ELISA. T cell responses were evaluated usingpooled splenocyte cultures (4 mice/group/time point). Antibody titerswere determined for individual mice (4 mice/group/time point).

TABLE 1 Vaccination Strategy Groups: A. Non-immune B. rWT1 + SE-VehicleC. rWT1 + MPL ®-SE D. 1°: WT1-DNA, 2°: WT1-DNA, 3°: WT1-Adeno E.rF-WT1/TRICOM ® F. 1°: rV-WT1/TRICOM ®, 2°: rF-WT1/TRICOM ®, 3°: rF-WT1/TRICOM ® Dose: rWT1: 50 ug MPL ®-SE: 10 ug WT1-DNA: 100 ugWT1-Adeno: 5 × 10⁵ pfu rF-WT1/TRICOM ®: 1 × 10⁸ pfu rV-WT1/TRICOM ®: 1 ×10⁸ pfu Route: Subcutaneous (200 ul) immunization for protein/adjuvantand vectors. Intramuscular immunization (50 ul) for WT1-DNA.

The full-length cDNA of the splice variant of WT1 used in these studiesis set forth in SEQ ID NO:381. The WT1-adenovirus used herein is asdescribed in Example 20. The rF-WT1/TRICOM® recombinant fowlpox and therV-WT1/TRICOM® recombinant vaccinia vectors both expressing WT1 andcontaining a triad of costimulatory molecules (B7-1, ICAM-1 and LFA-3)were generated by Therion Biologics (Cambridge, Mass., USA).

To evaluate T cell responses, splenocytes were stimulated in vitro withWT1 peptide “p32” (ARMFPNAPYLPSCLE [SEQ ID NO:509], amino acids 125-139of WT-1; found within the p117-139 peptide set forth in SEQ ID NO:2)known to contain a CTL and a helper T cell epitope. Intracellularcytokine staining for IFN-γ of p125-139 activated splenocytes at 21 daysfollowing the secondary immunization showed a significant percentage WT1responsive CD4⁺ and CD8⁺ T cells in mice immunized with rV-WT1/TRICOM®(prime) and rF-WT1/TRICOM® (boost) whereas no other groups showedsignificant WT1 specific T cell immune responses. Note that the DNAgroup was primed and boosted with DNA only. The same group subsequentlyhad an rWT1-Adv tertiary immunization.

Following tertiary immunization, responses to pools of overlapping

15-mer peptides were evaluated rather than responses to the singlepeptide #32. WT1 peptide CD8⁺ and CD4⁺ T-cells specific for peptide pool#32-36 were found to respond at levels similar to the responsesfollowing secondary immunization in mice vaccinated with rV-WT1/TRICOM®(prime) and rF-WT1/TRICOM® (boost X2). In addition, CD4⁺ and CD8⁺T-cells from these mice were found to respond, albeit at a much lowerlevel than peptide #32, to a second WT1 epitope contained within twooverlapping 15-mer peptides #57-58 (Peptide 57: DNLYQMTSQLECMTWN (aminoacids 224-239; SEQ ID NO:510); Peptide 58: MTSQLECMTWNQMNL (amino acids229-243; SEQ ID NO:511; overlap corresponds to amino acid residues224-243 of WT1).

Antibody responses were evaluated using a standard ELISA. Low levels ofserum IgG_(2b) antibodies to WT1 were measurable in all 8 mice 21 dayspost secondary immunization (titer of 1:1350) and 21 days post tertiaryimmunization (titer of 1:3325) in mice immunized with rWT1+MPL®-SE. Bycontrast, in all other groups serum IgG_(2b) antibodies titers were<1:100 (Table 2).

TABLE 2 Antibody Responses in WT1-Immunized Mice Serum Titers* SecondaryTertiary Group IgG₁ IgG_(2b) IgG₁ IgG_(2b) Non-immune ND ND ND ND WT1 +Vehicle-SE <75 <100 475  <50 WT1 + MPL ®-SE <50 1350 338 3325 WT1-DNA,WT1-DNA, ND ND ND ND WT1-Adeno rF-WT1/TRICOM ® ND ND <50  <50rV-WT/TRICOM ®, ND  <50 <50 ND rFWT1/TRICOM ® *Serum titers are theaverage of 4 mice per group. Titers written as <50, <75, or <100 werealso averages, but in all instances less than 4 mice had detectabletiters. ND indicates antibody was not detected in any of the mice.

In summary, immunization of C57BI/6 mice with rV-WT1/TRICOM® (prime)followed by rF-WT1/TRICOM® (boost) or by WT1-DNA (prime) followed byWT1-Adeno (boost) elicited both CD8 and CD4 T cell responses againstWT1. The T cell responses to rV-WT1/TRICOM® followed by rF-WT1/TRICOM®versus WT1-DNA followed by WT1-Adeno were equivalent within the power ofthe experimental design. Without being bound by theory, a majoradvantage of rF-WT1/TRICOM® is that multiple boosts can continue toincrease the level of immunity. Thus, these studies further confirm theimmunogenicity of the WT1 protein and provide support for the use of WT1DNA/adenovirus or rv-WT1/TRICOM®/rF-WT1/TRICOM® immunization regimens invaccine strategies for treating cancers associated with WT1 expression.

Example 38 Construction of the Stumpy-WT1-F Vector for Expression ofWT1-F in E. Coli

This example describes the construction of an expression vectorcontaining a truncated twin arginine translocator (TAT) signal peptidefused to the WT1-F reading frame (2-281 N-terminal portion of the WT1protein). This vector can be used to produce a single species truncatedTAT-WT1-F protein for use in immunization strategies for the treatmentof malignancies associated with expression of WT1.

As described previously in Example 34, the TAT signal sequence was usedto make various WT1 vectors. When these TAT vectors were used inexpression, multiple forms of the protein were observed. N-terminalsequencing of these forms showed that each of the three separateproteins being expressed were truncations of the TAT peptide. Thesecleavages were occurring at each of the twin arginine sites. Thus, atruncated TAT vector was constructed to shorten the TAT signal peptidefrom 39 amino acids to 12 amino acids to avoid generation of thesecleavage products during expression. The TAT “Stumpy” vector wasgenerated by maintaining the first 12 residues of the TAT signal peptideup to the first twin arginine. This vector was constructed as follows:

The following pairs of oligonucleotides were combined and then annealed.

Pair 1: Stumpy 1 (SEQ ID NO: 486): 5′ tatgaacaataacgatctgtttcaggc 3′Stumpy 3 (SEQ ID NO: 487): 5′ CTGAAACAGATCGTTATTGTTCA 3′ Pair 2: Stumpy4 (SEQ ID NO: 488): 5′ aattcttggtcattcacgtggcggctcgc 3′ Stumpy 2 (SEQ IDNO: 489): 5′ gagccgccacgtgaatgaccaag 3′

The pairs were then ligated together with pPDM, a modified pET28 vector,that had been digested with NdeI and EcoRI. The resulting plasmid,pStumpy, is set forth in SEQ ID NO:471. The amino acid sequence of thetruncated TAT protein is set forth in SEQ ID NO:506. The full-length TATpolynucleotide is set forth in SEQ ID NO:503 and encodes the amino acidsequence of the TAT polypeptide set forth in SEQ ID NO:504.

The pStumpy vector was then used to construct the Stumpy-WT1-F vector toexpress the N-terminal portion of WT1 without cleavage products. TheStumpy-WT1-F vector was constructed as follows:

The coding region of WT1-F was PCR amplified with the following primerset:

PDM-1005 (SEQ ID NO: 484): 5′ GGCTCCGACGTTCGGGACCTGAACGCACTG 3′ PDM-1004(SEQ ID NO: 485): 5′ CTGCAGAATTCATCACTGAATGCCTCTGAAG 3′

The amplification reaction contained 10 ul 10×Pfu buffer, 1 ul 10 mMdNTPs, 2 ul each 10 uM primer, 83 ul sterile water, and 1.5 ul Pfu DNApolymerase (Stratagene, La Jolla, Calif.). The reaction was firstdenatured for 2 minutes at 96° C. followed by 40 cycles of 96° C. for 20seconds, 63° C. for 15 seconds, and 72° C. for 30 seconds. The reactionwas then extended for a final extension of 72° C. for 4 minutes.

The PCR product was then digested with EcoRI and cloned into the pStumpyvector that had been digested with Eco72I and EcoRI. The construct wasconfirmed through sequence analysis and then transformed into BLR (DE3)pLys S cells. The resulting truncated TAT WT1-F fusion protein expressedas a single species. The polynucleotide sequence of the coding region ofpStumpy WT1-F is set forth in SEQ ID NO:469 which encodes the amino acidsequence set forth in SEQ ID NO:470.

Example 39 Construction of WT1-F GMP, WT1-G, and WT1-Delta G Vectors forExpression in E. Coli

This example describes the construction of WT1-F, WT1-G, and WT1-Delta Gvectors optimized for protein production in E. coli.

The TAT WT-1 F DNA sequence was codon optimized and syntheticallyengineered by Blue Heron Biotechnology (Bothel, Wash.) to be optimizedfor expression in E. coli. This codon optimized cDNA sequence of WT1-Fis set forth in SEQ ID NOs:477 and encodes the amino acid sequence setforth in SEQ ID NO:479. The plasmid containing this sequence was thendigested with NdeI and EcoRI and sub-cloned into a modified pET28 vectorthat had been digested with NdeI and EcoRI for expression in E. coli.This construct was confirmed to be correct by DNA sequence analysis andthen transformed into BLR (DE3) pLys S cells for expression. Thisconstruct is a purely synthetic traceable source of TAT WT1-F templateDNA.

The codon optimized sequence was then used as template for PCR to deletea proline rich region of fourteen amino acids from the N-terminal region(deletion of amino acids 54-67 of WT-1 F sequence set forth in SEQ IDNO:461; corresponding to amino acids 55-68 of full-length WT1), creatingthe WT-1 G cDNA sequence set forth in SEQ ID NO:473 which encodes theamino acid sequence set forth in SEQ ID NO:478. This was done by firstgenerating the pTAT-WT-1 G construct as follows:

The 5′ coding region of WT-1 G was PCR amplified using the followingprimer set:

PDM-1007 (SEQ ID NO: 490) 5′ caagcgctcatgagcccgaagtggcgagccc 3′ Tm76° C. PDM-1006 (SEQ ID NO: 491) 5′ cataaatttgcccgggcccgcccagagagccg3′ Tm 76° C.

The 3′ coding region of the WT-1 G region was PCR amplified using thefollowing primer set:

PDM-1008 (SEQ ID NO: 492) 5′ cattcattcatcaaacaggagcc 3′ Tm 61° C.PDM-1009 (SEQ ID NO: 493) 5′ ccattaagaattcatcactgaatacc 3′ Tm 60° C.

The PCR reaction for amplification of WT1-G contained 10 ul 10×Pfubuffer, 1 ul 10 mM dNTPs, 2 ul each 10 uM primer, 83 ul sterile water,1.5 ul Pfu DNA polymerase (Stratagene, La Jolla, Calif.), and 50 ng DNAtemplate (pTAT WT1-F codon optimized as set forth in SEQ ID NO: 477).The reaction was first denatured for 2 minutes at 96° C. followed by 40cycles of 96° C. for 20 seconds, 63° C. for 15 seconds, and 72° C. for30 seconds. The reaction was then extended for a final extension of 72°C. for 4 minutes.

The first PCR product was then digested with XbaI and cloned into pPDM(a modified pET28 vector) that had been digested with XbaI and Eco72I.The resulting construct (pTAT WT1-GA) was then digested with SrfI andEcoRI. The second PCR construct was digested with EcoRI and cloned intothe pTAT WT1-GA plasmid construct. At the same time a three-way ligationwas carried out by digesting the first PCR construct with XbaI and SrfIand digesting the second PCR construct with EcoRI and cloning into pPDMthat had been digested with XbaI and EcoRI. The resulting pTAT-WT1-Gconstruct was confirmed to be correct through sequence analysis and thentransformed into an expression host. The cDNA of pTAT-WT1-G is set forthin SEQ ID NO:475 and encodes the amino acid sequence of SEQ ID NO:482.

Due to incomplete SrfI digestion during the second method describedabove, an unexpected form of WT-1 G (WT-1 Delta G was created thatdeleted 14 amino acids (amino acids 55-68 of WT-1 F) and added threeadditional amino acids (WT-1 Delta G polynucleotide sequence set forthin SEQ ID NO:505 which encodes the polypeptide sequence set forth in SEQID NO:502). This TAT WT-1 Delta G construct resulted in an unexpected5-fold over expression as compared to the TAT WT-1 F constructs. ThecDNA of the pTAT-WT1 Delta G construct is set forth in SEQ ID NO:476 andencodes the amino acid sequence set forth in SEQ ID NO:481.

Following generation of the pTAT-WT-1 G, pStumpy-WT1 G and WT1-G withand without a His tag were constructed using the pTAT-WT1 G as atemplate in the PCR reactions described below.

The coding region of the WT1-G region was PCR amplified using thefollowing primer sets:

PCR 1: PDM-1010 (SEQ ID NO: 494) 5′ ggttcggatgtacgcgatctgaacg 3′ Tm68° C. PDM-1011 (SEQ ID NO: 495) 5′ caaagaattcatcactgaataccgcg 3′ Tm63° C. PCR 2: PDM-1023 (SEQ ID NO: 496)5′ gcttttggcatatgggttcggatgtacgcgatc 3′ Tm 71° C. PDM-1011 (SEQ ID NO:497) 5′ caaagaattcatcactgaataccgcg 3′ Tm 63° C.

The PCR reaction and amplification conditions were as described aboveusing the pTAT-WT1 G set forth in SEQ ID NO:475 as template. The firstPCR product was digested with EcoRI and cloned into pPDM (a modifiedpET28 vector with a his tag in-frame) and pSTUMPY (a modified pET28vector with a 12 amino acid truncated TAT leader peptide in frame setforth in SEQ ID NO:471) that were digested with EcoRI and Eco72I. Thesecond PCR product was digested with NdeI and EcoRI and cloned into amodified pET28 vector (pPDM) that had been digested with NdeI and EcoRI.The constructs were confirmed to be correct through sequence analysisand then transformed into an expression host. The resulting constructsare as follows: The polynucleotide of WT1-G with His tag is set forth inSEQ ID NO:472 and encodes the amino acid sequence set forth in SEQ IDNO:480. The polynucleotide of WT1-G without His tag is set forth in SEQID NO:473 which encodes the amino acid sequence set forth in SEQ IDNO:478. The pStumpy-WT1-G polynucleotide sequence is set forth in SEQ IDNO:474 and encodes the amino acid sequence set forth in SEQ ID NO:483.

In a related experiment, the pStumpy WT-1 F GMP (codon optimized) vectorwas constructed as follows:

The coding region of the WT-1 F region was amplified using the followingprimers

PDM-1010 (SEQ ID NO: 500) 5′ ggttcggatgtacgcgatctgaacg 3′ Tm 68° C.PDM-1011 (SEQ ID NO: 501) 5′ caaagaattcatcactgaataccgcg 3′ Tm 63° C.

The PCR reaction and amplification conditions were as described aboveusing pTAT WT-1F GMP as template. The PCR product was digested withEcoRI and cloned into the pStumpy vector described in Example 38 thatwas digested with EcoRI and Eco72I. The construct was confirmed to becorrect through sequence analysis and then transformed into anexpression host. The polynucleotide sequence of pStumpy WT-1 F GMP isset forth in SEQ ID NO:498 which encodes the amino acid sequence of SEQID NO:499.

In summary, the expression vectors described herein can be used foroptimal expression and production of WT1, truncated WT1, and WT1 fusionproteins for use in vaccine strategies for the treatment of malignanciesassociated with expression of WT1.

Example 40 Construction of an Adenovirus Vector Expressing Human WT1-F

WT1-F including a start codon (the DNA sequence of WT1-F including thestart codon is set forth in SEQ ID NO:508) was cloned into an E1 and E3deleted adenovirus serotype 5 vector. The resulting adenovirus vector isset forth in SEQ ID NO:507. The expression of WT1-F is controlled by theCMV promoter mediating high levels of WT1-F protein expression.Infection of human cells with this reagent leads to a high level ofexpression of the WT1-F protein. The antigenic nature of the adenoviralproteins introduced into the host cell during and produced at low levelssubsequent to infection can act to increase immune surveillance andimmune recognition of WT1 as an immunological target. This vector can bealso used to generate immune responses against the WT1 protein wheninnoculated into mammalian subjects. If these subjects are positive forWT1 expressing tumor cells the immune response could have a therapeuticor curative effect on the course of the disease.

Example 41 CTL Responses in HLA-A2 Transgenic Mice WITH WT-1 ProteinPlus Various Adjuvants

Groups of four HLA-A2/Kb transgenic mice were immunized intradermallythree times at three week intervals with 20 μg of WT1-F truncationprotein (see Example 35) admixed with one of the following adjuvants, 10μg RC-529™-SE, 10 μg ENHANZYN®, or 10 μg RC-529™-AF+10 μg QuilA. Apositive control group were immunized twice with WT1-F DNA followed by aboost with 10⁷ PFU adenovirus encoding WT1-F (see example 40). Mice weresacrificed 2 weeks after the final immunization and CTL were measured.Spleens were harvested and stimulated in vitro with CD.24-A2Kb-WT-1-LAMP cells (see Example 18) and assayed against Jurkat A2 Kbcells expressing full length WT1-F or pulsed with WT-1 CD8 epitopepeptides (p10-18 (ALLPAVPSL, SEQ ID NO:451), p32 (ARMFPNAPYLPSCLE [SEQID NO:509], aa 125-139 found within p117-139 SEQ ID NO:2, and WT1 p37-45peptide (VLDFAPPGA, human WT1 residues 37-45; SEQ ID NO:241). A standardchromium release assay was used to assay for CTL activity. As observedin previous experiments, immunization with WT1 DNA followed byadenoviral boost elicited a WT1-specific CTL response in HLA-A2transgenic mice. CTL responses were also detected from the spleens ofmice immunized with WT1-F protein in combination with ENHANZYN®,RC-529™-SE and the combination of RC-529™-AF and QuilA. The magnitude ofthe responses induced was comparable to that seen in the DNA-Adenovirusimmunization.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1.-11. (canceled)
 12. An isolated polynucleotide encoding a polypeptidecomprising an amino acid sequence selected from SEQ ID Nos:470, 479,480, 481, 482, and
 483. 13. The isolated polynucleotide of claim 12wherein the polynucleotide has been codon optimized for expression in E.coli.
 14. The isolated polynucleotide of claim 12 wherein thepolynucleotide comprises a sequence selected from the group consistingof SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:472, SEQ ID NO:473, SEQ IDNO:474, SEQ ID NO:475, SEQ ID NO:476, SEQ ID NO:477, SEQ ID NO:503, andSEQ ID NO:505.
 15. A composition comprising an isolated polynucleotideof claim 12 and a pharmaceutically acceptable carrier or excipient.16.-18. (canceled)
 19. An expression vector comprising a polynucleotideof claim 12 operably linked to an expression control sequence.
 20. Ahost cell transformed or transfected with an expression vector accordingto claim
 19. 21. An isolated polynucleotide encoding a polypeptidecomprising SEQ ID NO:470.
 22. An isolated polynucleotide according toclaim 21 wherein the polynucleotide has been codon optimized forexpression in E. coli.
 23. An isolated polynucleotide according to claim21 wherein the polynucleotide comprises a sequence selected from thegroup consisting of SEQ ID NO:469 and SEQ ID NO:471.
 24. A compositioncomprising an isolated polynucleotide according to claim 21 and apharmaceutically acceptable carrier or excipient.
 25. An expressionvector comprising a polynucleotide according to claim 21 operably linkedto an expression control sequence.
 26. A host cell transformed ortransfected with an expression vector according to claim
 25. 27. Anisolated polynucleotide encoding a polypeptide comprising SEQ ID NO:479.28. An isolated polynucleotide according to claim 27 wherein thepolynucleotide has been codon optimized for expression in E. coli. 29.An isolated polynucleotide according to claim 27 wherein thepolynucleotide comprises SEQ ID NO:477.
 30. A composition comprising anisolated polynucleotide according to claim 27 and a pharmaceuticallyacceptable carrier or excipient.
 31. An expression vector comprising apolynucleotide according to claim 27 operably linked to an expressioncontrol sequence.
 32. A host cell transformed or transfected with anexpression vector according to claim 31.